US20240066126A1 - Combination therapy of solid tumors using chimeric antigen receptor cells representing adaptive and innate immunity - Google Patents
Combination therapy of solid tumors using chimeric antigen receptor cells representing adaptive and innate immunity Download PDFInfo
- Publication number
- US20240066126A1 US20240066126A1 US18/455,544 US202318455544A US2024066126A1 US 20240066126 A1 US20240066126 A1 US 20240066126A1 US 202318455544 A US202318455544 A US 202318455544A US 2024066126 A1 US2024066126 A1 US 2024066126A1
- Authority
- US
- United States
- Prior art keywords
- cells
- car
- methods include
- preferred methods
- domain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 141
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 title claims abstract description 89
- 238000002648 combination therapy Methods 0.000 title abstract description 3
- 230000004721 adaptive immunity Effects 0.000 title description 4
- 230000015788 innate immune response Effects 0.000 title description 2
- 210000003370 receptor cell Anatomy 0.000 title description 2
- 210000004027 cell Anatomy 0.000 claims abstract description 140
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 85
- 201000011510 cancer Diseases 0.000 claims abstract description 32
- 230000000295 complement effect Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 275
- 239000000427 antigen Substances 0.000 claims description 88
- 108091007433 antigens Proteins 0.000 claims description 84
- 102000036639 antigens Human genes 0.000 claims description 84
- 210000002540 macrophage Anatomy 0.000 claims description 72
- 230000004068 intracellular signaling Effects 0.000 claims description 59
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 claims description 34
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 claims description 34
- 210000004443 dendritic cell Anatomy 0.000 claims description 29
- 230000003834 intracellular effect Effects 0.000 claims description 20
- 210000001616 monocyte Anatomy 0.000 claims description 20
- 210000002865 immune cell Anatomy 0.000 claims description 19
- 108091033319 polynucleotide Proteins 0.000 claims description 16
- 102000040430 polynucleotide Human genes 0.000 claims description 16
- 210000004369 blood Anatomy 0.000 claims description 14
- 239000008280 blood Substances 0.000 claims description 14
- 230000001939 inductive effect Effects 0.000 claims description 14
- 230000004614 tumor growth Effects 0.000 claims description 12
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims description 10
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 claims description 10
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 claims description 10
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 8
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 8
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 8
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 claims description 7
- 102100039360 Toll-like receptor 4 Human genes 0.000 claims description 7
- 108020004999 messenger RNA Proteins 0.000 claims description 7
- 239000012636 effector Substances 0.000 claims description 6
- 101000595548 Homo sapiens TIR domain-containing adapter molecule 1 Proteins 0.000 claims description 5
- 102100036073 TIR domain-containing adapter molecule 1 Human genes 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 4
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 4
- 230000003044 adaptive effect Effects 0.000 claims description 3
- 230000005880 cancer cell killing Effects 0.000 claims description 3
- 210000003690 classically activated macrophage Anatomy 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 3
- 230000000242 pagocytic effect Effects 0.000 claims description 2
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 claims 1
- 238000011282 treatment Methods 0.000 abstract description 14
- 238000002512 chemotherapy Methods 0.000 abstract description 8
- 238000013459 approach Methods 0.000 abstract description 6
- 230000001900 immune effect Effects 0.000 abstract description 5
- 238000001959 radiotherapy Methods 0.000 abstract description 5
- 230000009467 reduction Effects 0.000 abstract description 3
- 239000002671 adjuvant Substances 0.000 abstract description 2
- 230000004048 modification Effects 0.000 abstract description 2
- 238000012986 modification Methods 0.000 abstract description 2
- 210000005006 adaptive immune system Anatomy 0.000 abstract 1
- 230000004075 alteration Effects 0.000 abstract 1
- 230000004888 barrier function Effects 0.000 abstract 1
- 230000001024 immunotherapeutic effect Effects 0.000 abstract 1
- 238000009169 immunotherapy Methods 0.000 abstract 1
- 210000005007 innate immune system Anatomy 0.000 abstract 1
- 210000000822 natural killer cell Anatomy 0.000 description 44
- 108090000623 proteins and genes Proteins 0.000 description 39
- 210000004881 tumor cell Anatomy 0.000 description 32
- 108090000765 processed proteins & peptides Proteins 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 24
- 102000005962 receptors Human genes 0.000 description 20
- 108020003175 receptors Proteins 0.000 description 20
- 239000012634 fragment Substances 0.000 description 17
- 102100026890 Tumor necrosis factor ligand superfamily member 4 Human genes 0.000 description 15
- 230000014509 gene expression Effects 0.000 description 15
- 102000004196 processed proteins & peptides Human genes 0.000 description 15
- 108010042215 OX40 Ligand Proteins 0.000 description 14
- 239000003795 chemical substances by application Substances 0.000 description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 description 13
- 230000004913 activation Effects 0.000 description 13
- 108010002350 Interleukin-2 Proteins 0.000 description 12
- 102000000588 Interleukin-2 Human genes 0.000 description 12
- 230000011664 signaling Effects 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 150000007523 nucleic acids Chemical class 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 206010025323 Lymphomas Diseases 0.000 description 9
- 230000000735 allogeneic effect Effects 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 230000028993 immune response Effects 0.000 description 9
- 230000037449 immunogenic cell death Effects 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 239000003446 ligand Substances 0.000 description 9
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 9
- 241000124008 Mammalia Species 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 230000030833 cell death Effects 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 125000006850 spacer group Chemical group 0.000 description 8
- 230000008685 targeting Effects 0.000 description 8
- 241001430294 unidentified retrovirus Species 0.000 description 8
- 102000004082 Calreticulin Human genes 0.000 description 7
- 108090000549 Calreticulin Proteins 0.000 description 7
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 7
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 7
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 7
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 7
- 206010057249 Phagocytosis Diseases 0.000 description 7
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 7
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 230000008782 phagocytosis Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 239000013603 viral vector Substances 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 description 6
- 102000013691 Interleukin-17 Human genes 0.000 description 6
- 108050003558 Interleukin-17 Proteins 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 102000002689 Toll-like receptor Human genes 0.000 description 6
- 108020000411 Toll-like receptor Proteins 0.000 description 6
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 6
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 6
- 230000001093 anti-cancer Effects 0.000 description 6
- 210000000612 antigen-presenting cell Anatomy 0.000 description 6
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 6
- 230000007423 decrease Effects 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 230000002163 immunogen Effects 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 230000002062 proliferating effect Effects 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 description 5
- 101000809875 Homo sapiens TYRO protein tyrosine kinase-binding protein Proteins 0.000 description 5
- 102100036678 Interleukin-27 subunit alpha Human genes 0.000 description 5
- 241000713666 Lentivirus Species 0.000 description 5
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 description 5
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 102100038717 TYRO protein tyrosine kinase-binding protein Human genes 0.000 description 5
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 5
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 230000001086 cytosolic effect Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 235000020887 ketogenic diet Nutrition 0.000 description 5
- 230000002147 killing effect Effects 0.000 description 5
- 239000002157 polynucleotide Substances 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 5
- 101150047061 tag-72 gene Proteins 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 208000026310 Breast neoplasm Diseases 0.000 description 4
- 102100028681 C-type lectin domain family 4 member K Human genes 0.000 description 4
- 108010087819 Fc receptors Proteins 0.000 description 4
- 102000009109 Fc receptors Human genes 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 102100034671 L-lactate dehydrogenase A chain Human genes 0.000 description 4
- 108010088350 Lactate Dehydrogenase 5 Proteins 0.000 description 4
- 206010027476 Metastases Diseases 0.000 description 4
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 4
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 230000001640 apoptogenic effect Effects 0.000 description 4
- 239000011230 binding agent Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 4
- 210000003038 endothelium Anatomy 0.000 description 4
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 239000002955 immunomodulating agent Substances 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 230000002601 intratumoral effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 210000004985 myeloid-derived suppressor cell Anatomy 0.000 description 4
- 230000037452 priming Effects 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 4
- 238000010361 transduction Methods 0.000 description 4
- 230000026683 transduction Effects 0.000 description 4
- 210000004981 tumor-associated macrophage Anatomy 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 3
- 102100031151 C-C chemokine receptor type 2 Human genes 0.000 description 3
- 101710183165 C-type lectin domain family 4 member K Proteins 0.000 description 3
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 3
- 101150029707 ERBB2 gene Proteins 0.000 description 3
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 description 3
- 101000831496 Homo sapiens Toll-like receptor 3 Proteins 0.000 description 3
- 101000669460 Homo sapiens Toll-like receptor 5 Proteins 0.000 description 3
- 101000669402 Homo sapiens Toll-like receptor 7 Proteins 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 102000003814 Interleukin-10 Human genes 0.000 description 3
- 108090000174 Interleukin-10 Proteins 0.000 description 3
- 108010065805 Interleukin-12 Proteins 0.000 description 3
- 102000013462 Interleukin-12 Human genes 0.000 description 3
- 102000003812 Interleukin-15 Human genes 0.000 description 3
- 108090000172 Interleukin-15 Proteins 0.000 description 3
- 108010063954 Mucins Proteins 0.000 description 3
- 102000015728 Mucins Human genes 0.000 description 3
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- 102100021923 Prolow-density lipoprotein receptor-related protein 1 Human genes 0.000 description 3
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 3
- 101710100968 Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 3
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 3
- 108091027967 Small hairpin RNA Proteins 0.000 description 3
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 3
- 102100024333 Toll-like receptor 2 Human genes 0.000 description 3
- 102100024324 Toll-like receptor 3 Human genes 0.000 description 3
- 102100039357 Toll-like receptor 5 Human genes 0.000 description 3
- 102100039390 Toll-like receptor 7 Human genes 0.000 description 3
- 102100033117 Toll-like receptor 9 Human genes 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 102100040247 Tumor necrosis factor Human genes 0.000 description 3
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 108700010039 chimeric receptor Proteins 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000001461 cytolytic effect Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 230000003511 endothelial effect Effects 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 230000036210 malignancy Effects 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000001177 retroviral effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000004055 small Interfering RNA Substances 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 230000000381 tumorigenic effect Effects 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- 101100339431 Arabidopsis thaliana HMGB2 gene Proteins 0.000 description 2
- 108091008875 B cell receptors Proteins 0.000 description 2
- 101710149815 C-C chemokine receptor type 2 Proteins 0.000 description 2
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 2
- 102100038078 CD276 antigen Human genes 0.000 description 2
- 108010029697 CD40 Ligand Proteins 0.000 description 2
- 101150013553 CD40 gene Proteins 0.000 description 2
- 102100032937 CD40 ligand Human genes 0.000 description 2
- 102100022002 CD59 glycoprotein Human genes 0.000 description 2
- 102100035793 CD83 antigen Human genes 0.000 description 2
- 108090000835 CX3C Chemokine Receptor 1 Proteins 0.000 description 2
- 102100039196 CX3C chemokine receptor 1 Human genes 0.000 description 2
- 201000009030 Carcinoma Diseases 0.000 description 2
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 2
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 108010039471 Fas Ligand Protein Proteins 0.000 description 2
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 2
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 2
- 108060005986 Granzyme Proteins 0.000 description 2
- 102000001398 Granzyme Human genes 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 108700010013 HMGB1 Proteins 0.000 description 2
- 101150021904 HMGB1 gene Proteins 0.000 description 2
- 102100029360 Hematopoietic cell signal transducer Human genes 0.000 description 2
- 102100037907 High mobility group protein B1 Human genes 0.000 description 2
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 2
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 2
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 2
- 101000990188 Homo sapiens Hematopoietic cell signal transducer Proteins 0.000 description 2
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 2
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 2
- 101001134216 Homo sapiens Macrophage scavenger receptor types I and II Proteins 0.000 description 2
- 206010020843 Hyperthermia Diseases 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 108010047761 Interferon-alpha Proteins 0.000 description 2
- 102000006992 Interferon-alpha Human genes 0.000 description 2
- 102000003777 Interleukin-1 beta Human genes 0.000 description 2
- 108090000193 Interleukin-1 beta Proteins 0.000 description 2
- 102000004551 Interleukin-10 Receptors Human genes 0.000 description 2
- 108010017550 Interleukin-10 Receptors Proteins 0.000 description 2
- 102000004560 Interleukin-12 Receptors Human genes 0.000 description 2
- 108010017515 Interleukin-12 Receptors Proteins 0.000 description 2
- 108010017535 Interleukin-15 Receptors Proteins 0.000 description 2
- 102000004556 Interleukin-15 Receptors Human genes 0.000 description 2
- 102000003810 Interleukin-18 Human genes 0.000 description 2
- 108090000171 Interleukin-18 Proteins 0.000 description 2
- 102000004557 Interleukin-18 Receptors Human genes 0.000 description 2
- 108010017537 Interleukin-18 Receptors Proteins 0.000 description 2
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 2
- 102100036705 Interleukin-23 subunit alpha Human genes 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 108010038498 Interleukin-7 Receptors Proteins 0.000 description 2
- 102000010782 Interleukin-7 Receptors Human genes 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 description 2
- 239000013255 MILs Substances 0.000 description 2
- 102100034184 Macrophage scavenger receptor types I and II Human genes 0.000 description 2
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 description 2
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 description 2
- 108010004217 Natural Cytotoxicity Triggering Receptor 1 Proteins 0.000 description 2
- 102100032870 Natural cytotoxicity triggering receptor 1 Human genes 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 108060008245 Thrombospondin Proteins 0.000 description 2
- 102000002938 Thrombospondin Human genes 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 2
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000000556 agonist Substances 0.000 description 2
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000002491 angiogenic effect Effects 0.000 description 2
- 229940045799 anthracyclines and related substance Drugs 0.000 description 2
- 230000002494 anti-cea effect Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000003190 augmentative effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000016396 cytokine production Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 230000004041 dendritic cell maturation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 229960003638 dopamine Drugs 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 102000054766 genetic haplotypes Human genes 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 230000036031 hyperthermia Effects 0.000 description 2
- 230000006028 immune-suppresssive effect Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 2
- 210000000066 myeloid cell Anatomy 0.000 description 2
- 210000000581 natural killer T-cell Anatomy 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000010287 polarization Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229940126622 therapeutic monoclonal antibody Drugs 0.000 description 2
- 230000002463 transducing effect Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000005747 tumor angiogenesis Effects 0.000 description 2
- 231100000588 tumorigenic Toxicity 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 229960003048 vinblastine Drugs 0.000 description 2
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 2
- DMWMUMWKGKGSNW-OPMCLZTFSA-N (2S)-6-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-4-amino-2-[[2-[[(2R)-2-amino-3-[(2R)-2,3-di(hexadecanoyloxy)propyl]sulfanylpropanoyl]amino]acetyl]amino]-4-oxobutanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-hydroxypropanoyl]amino]-4-oxobutanoyl]amino]-3-methylpentanoyl]amino]-3-hydroxypropanoyl]amino]-3-phenylpropanoyl]amino]hexanoyl]amino]-4-carboxybutanoyl]amino]hexanoic acid Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](CSC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(O)=O)OC(=O)CCCCCCCCCCCCCCC DMWMUMWKGKGSNW-OPMCLZTFSA-N 0.000 description 1
- OEDPHAKKZGDBEV-GFPBKZJXSA-N (2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2r)-3-[2,3-di(hexadecanoyloxy)propylsulfanyl]-2-(hexadecanoylamino)propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoic acid Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)CCCCCCCCCCCCCCC)CSCC(COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC OEDPHAKKZGDBEV-GFPBKZJXSA-N 0.000 description 1
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- FHJATBIERQTCTN-UHFFFAOYSA-N 1-[4-amino-2-(ethylaminomethyl)imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol Chemical compound C1=CC=CC2=C(N(C(CNCC)=N3)CC(C)(C)O)C3=C(N)N=C21 FHJATBIERQTCTN-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 108010082808 4-1BB Ligand Proteins 0.000 description 1
- 102000002627 4-1BB Ligand Human genes 0.000 description 1
- 102100023990 60S ribosomal protein L17 Human genes 0.000 description 1
- 208000010444 Acidosis Diseases 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 108010029445 Agammaglobulinaemia Tyrosine Kinase Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 108010083359 Antigen Receptors Proteins 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- 102000004452 Arginase Human genes 0.000 description 1
- 102100021723 Arginase-1 Human genes 0.000 description 1
- 101710129000 Arginase-1 Proteins 0.000 description 1
- 108700024123 Arginases Proteins 0.000 description 1
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 102100021663 Baculoviral IAP repeat-containing protein 5 Human genes 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 102100023701 C-C motif chemokine 18 Human genes 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 102100038077 CD226 antigen Human genes 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 108010055167 CD59 Antigens Proteins 0.000 description 1
- 102100025221 CD70 antigen Human genes 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 241001227713 Chiron Species 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 102100025877 Complement component C1q receptor Human genes 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 101100016370 Danio rerio hsp90a.1 gene Proteins 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 101100285708 Dictyostelium discoideum hspD gene Proteins 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 1
- 238000011510 Elispot assay Methods 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101000740462 Escherichia coli Beta-lactamase TEM Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 108010040721 Flagellin Proteins 0.000 description 1
- 102100021260 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Human genes 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102100028967 HLA class I histocompatibility antigen, alpha chain G Human genes 0.000 description 1
- 102100036242 HLA class II histocompatibility antigen, DQ alpha 2 chain Human genes 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 108010024164 HLA-G Antigens Proteins 0.000 description 1
- 206010066476 Haematological malignancy Diseases 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 102100022132 High affinity immunoglobulin epsilon receptor subunit gamma Human genes 0.000 description 1
- 108091010847 High affinity immunoglobulin epsilon receptor subunit gamma Proteins 0.000 description 1
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000777599 Homo sapiens C-C chemokine receptor type 2 Proteins 0.000 description 1
- 101000978371 Homo sapiens C-C motif chemokine 18 Proteins 0.000 description 1
- 101000884298 Homo sapiens CD226 antigen Proteins 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101000897400 Homo sapiens CD59 glycoprotein Proteins 0.000 description 1
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 1
- 101000933665 Homo sapiens Complement component C1q receptor Proteins 0.000 description 1
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 1
- 101000894906 Homo sapiens Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Proteins 0.000 description 1
- 101000930801 Homo sapiens HLA class II histocompatibility antigen, DQ alpha 2 chain Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 1
- 101001046870 Homo sapiens Hypoxia-inducible factor 1-alpha Proteins 0.000 description 1
- 101000878602 Homo sapiens Immunoglobulin alpha Fc receptor Proteins 0.000 description 1
- 101000971538 Homo sapiens Killer cell lectin-like receptor subfamily F member 1 Proteins 0.000 description 1
- 101000971532 Homo sapiens Killer cell lectin-like receptor subfamily F member 2 Proteins 0.000 description 1
- 101000984189 Homo sapiens Leukocyte immunoglobulin-like receptor subfamily B member 2 Proteins 0.000 description 1
- 101000984186 Homo sapiens Leukocyte immunoglobulin-like receptor subfamily B member 4 Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 1
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 description 1
- 101000991061 Homo sapiens MHC class I polypeptide-related sequence B Proteins 0.000 description 1
- 101000916644 Homo sapiens Macrophage colony-stimulating factor 1 receptor Proteins 0.000 description 1
- 101001109503 Homo sapiens NKG2-C type II integral membrane protein Proteins 0.000 description 1
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 description 1
- 101000589305 Homo sapiens Natural cytotoxicity triggering receptor 2 Proteins 0.000 description 1
- 101000633516 Homo sapiens Nuclear receptor subfamily 2 group F member 6 Proteins 0.000 description 1
- 101001043564 Homo sapiens Prolow-density lipoprotein receptor-related protein 1 Proteins 0.000 description 1
- 101000663183 Homo sapiens Scavenger receptor class F member 1 Proteins 0.000 description 1
- 101000663187 Homo sapiens Scavenger receptor class F member 2 Proteins 0.000 description 1
- 101000984753 Homo sapiens Serine/threonine-protein kinase B-raf Proteins 0.000 description 1
- 101100207070 Homo sapiens TNFSF8 gene Proteins 0.000 description 1
- 101000597785 Homo sapiens Tumor necrosis factor receptor superfamily member 6B Proteins 0.000 description 1
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 1
- 101000850748 Homo sapiens Tumor necrosis factor receptor type 1-associated DEATH domain protein Proteins 0.000 description 1
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 description 1
- 102100022875 Hypoxia-inducible factor 1-alpha Human genes 0.000 description 1
- 108010073816 IgE Receptors Proteins 0.000 description 1
- 102000009438 IgE Receptors Human genes 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102100038005 Immunoglobulin alpha Fc receptor Human genes 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000004554 Interleukin-17 Receptors Human genes 0.000 description 1
- 108010017525 Interleukin-17 Receptors Proteins 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108010038452 Interleukin-3 Receptors Proteins 0.000 description 1
- 102000010790 Interleukin-3 Receptors Human genes 0.000 description 1
- 102000010787 Interleukin-4 Receptors Human genes 0.000 description 1
- 108010038486 Interleukin-4 Receptors Proteins 0.000 description 1
- 102100021592 Interleukin-7 Human genes 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 102100021593 Interleukin-7 receptor subunit alpha Human genes 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 102000000585 Interleukin-9 Human genes 0.000 description 1
- 102000010682 Interleukin-9 Receptors Human genes 0.000 description 1
- 108010038414 Interleukin-9 Receptors Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 108020003285 Isocitrate lyase Proteins 0.000 description 1
- 102100021458 Killer cell lectin-like receptor subfamily F member 1 Human genes 0.000 description 1
- 102100021456 Killer cell lectin-like receptor subfamily F member 2 Human genes 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 102100025583 Leukocyte immunoglobulin-like receptor subfamily B member 2 Human genes 0.000 description 1
- 102100025578 Leukocyte immunoglobulin-like receptor subfamily B member 4 Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108010015340 Low Density Lipoprotein Receptor-Related Protein-1 Proteins 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 102000018170 Lymphotoxin beta Receptor Human genes 0.000 description 1
- 108010091221 Lymphotoxin beta Receptor Proteins 0.000 description 1
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 1
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 102100030301 MHC class I polypeptide-related sequence A Human genes 0.000 description 1
- 102100030300 MHC class I polypeptide-related sequence B Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 108010058398 Macrophage Colony-Stimulating Factor Receptor Proteins 0.000 description 1
- 102100028198 Macrophage colony-stimulating factor 1 receptor Human genes 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 206010027406 Mesothelioma Diseases 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 1
- 101100481584 Mus musculus Tlr1 gene Proteins 0.000 description 1
- 101100207071 Mus musculus Tnfsf8 gene Proteins 0.000 description 1
- 102100022683 NKG2-C type II integral membrane protein Human genes 0.000 description 1
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 description 1
- 108010004222 Natural Cytotoxicity Triggering Receptor 3 Proteins 0.000 description 1
- 102100032851 Natural cytotoxicity triggering receptor 2 Human genes 0.000 description 1
- 102100032852 Natural cytotoxicity triggering receptor 3 Human genes 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 102100029528 Nuclear receptor subfamily 2 group F member 6 Human genes 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 102000004503 Perforin Human genes 0.000 description 1
- 108010056995 Perforin Proteins 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 1
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 1
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 208000007660 Residual Neoplasm Diseases 0.000 description 1
- 241000712907 Retroviridae Species 0.000 description 1
- 102100037081 Scavenger receptor class F member 1 Human genes 0.000 description 1
- 102100037076 Scavenger receptor class F member 2 Human genes 0.000 description 1
- 101100071627 Schizosaccharomyces pombe (strain 972 / ATCC 24843) swo1 gene Proteins 0.000 description 1
- 102100027103 Serine/threonine-protein kinase B-raf Human genes 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108010002687 Survivin Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 108091005735 TGF-beta receptors Proteins 0.000 description 1
- 210000000068 Th17 cell Anatomy 0.000 description 1
- 102000036693 Thrombopoietin Human genes 0.000 description 1
- 108010041111 Thrombopoietin Proteins 0.000 description 1
- 102000016715 Transforming Growth Factor beta Receptors Human genes 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 1
- 102100032100 Tumor necrosis factor ligand superfamily member 8 Human genes 0.000 description 1
- 102100035284 Tumor necrosis factor receptor superfamily member 6B Human genes 0.000 description 1
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 1
- 102100033081 Tumor necrosis factor receptor type 1-associated DEATH domain protein Human genes 0.000 description 1
- 108091005906 Type I transmembrane proteins Proteins 0.000 description 1
- 102100029823 Tyrosine-protein kinase BTK Human genes 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229920000392 Zymosan Polymers 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000007950 acidosis Effects 0.000 description 1
- 208000026545 acidosis disease Diseases 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 230000006536 aerobic glycolysis Effects 0.000 description 1
- 108700025316 aldesleukin Proteins 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 230000006023 anti-tumor response Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 238000002617 apheresis Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000002886 autophagic effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000000053 blastoma Diseases 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000000981 bystander Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- SQQXRXKYTKFFSM-UHFFFAOYSA-N chembl1992147 Chemical compound OC1=C(OC)C(OC)=CC=C1C1=C(C)C(C(O)=O)=NC(C=2N=C3C4=NC(C)(C)N=C4C(OC)=C(O)C3=CC=2)=C1N SQQXRXKYTKFFSM-UHFFFAOYSA-N 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 208000011654 childhood malignant neoplasm Diseases 0.000 description 1
- 208000012191 childhood neoplasm Diseases 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000779 depleting effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 210000003515 double negative t cell Anatomy 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 201000008184 embryoma Diseases 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 108700014844 flt3 ligand Proteins 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 description 1
- 229940124670 gardiquimod Drugs 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 101150086609 groEL2 gene Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 229940124669 imidazoquinoline Drugs 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000008102 immune modulation Effects 0.000 description 1
- 102000027596 immune receptors Human genes 0.000 description 1
- 108091008915 immune receptors Proteins 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000013394 immunophenotyping Methods 0.000 description 1
- 230000002480 immunoprotective effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 108010045648 interferon omega 1 Proteins 0.000 description 1
- 108700027921 interferon tau Proteins 0.000 description 1
- 229960001388 interferon-beta Drugs 0.000 description 1
- 229940076144 interleukin-10 Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 210000003738 lymphoid progenitor cell Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108010051618 macrophage stimulatory lipopeptide 2 Proteins 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 239000010445 mica Substances 0.000 description 1
- 229910052618 mica group Inorganic materials 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 210000004980 monocyte derived macrophage Anatomy 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000003643 myeloid progenitor cell Anatomy 0.000 description 1
- 210000004479 myeloid suppressor cell Anatomy 0.000 description 1
- 230000002410 myeloprotective effect Effects 0.000 description 1
- 210000004296 naive t lymphocyte Anatomy 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 244000309459 oncolytic virus Species 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 210000003668 pericyte Anatomy 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940087463 proleukin Drugs 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000001303 quality assessment method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000003439 radiotherapeutic effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 108010003189 recombinant human tumor necrosis factor-binding protein-1 Proteins 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 229950010550 resiquimod Drugs 0.000 description 1
- BXNMTOQRYBFHNZ-UHFFFAOYSA-N resiquimod Chemical compound C1=CC=CC2=C(N(C(COCC)=N3)CC(C)(C)O)C3=C(N)N=C21 BXNMTOQRYBFHNZ-UHFFFAOYSA-N 0.000 description 1
- 108010056030 retronectin Proteins 0.000 description 1
- KNUXHTWUIVMBBY-JRJYXWDASA-N rintatolimod Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1.O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1.O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(NC=NC2=O)=C2N=C1 KNUXHTWUIVMBBY-JRJYXWDASA-N 0.000 description 1
- 229950006564 rintatolimod Drugs 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000006807 siRNA silencing Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000003614 tolerogenic effect Effects 0.000 description 1
- 230000023750 transforming growth factor beta production Effects 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
- 238000013414 tumor xenograft model Methods 0.000 description 1
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 1
- 230000002476 tumorcidal effect Effects 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 230000004222 uncontrolled growth Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 210000000605 viral structure Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1725—Complement proteins, e.g. anaphylatoxin, C3a or C5a
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/193—Colony stimulating factors [CSF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4614—Monocytes; Macrophages
Definitions
- the teachings herein relate to the use of modified immunological cells for the treatment of cancer.
- mAbs therapeutic monoclonal antibodies
- CARs chimeric antigen receptors
- mAbs therapeutic monoclonal antibodies
- CARs chimeric antigen receptors
- Chimeric antigen receptors are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell.
- mAb monoclonal antibody
- Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus (binder), and a transmembrane domain connected to an endodomain which transmits T-cell activation signals.
- scFv single-chain variable fragments
- monoclonal antibodies which recognize a target antigen, fused via a trans-membrane domain to a signaling endodomain.
- T cells When T cells express such a CAR, they recognize and kill target cells that express the target antigen.
- CARs have been developed against various tumor-associated antigens and many are currently undergoing clinical trials. Although CAR-T cell-mediated treatment have shown success towards compact target antigens such as CD19 or GD2, chimeric antigen receptors often to fail to signal in response to antigens with bulky extracellular domains. There is therefore a need for alternative CAR T-cell approaches, capable of killing target cells expressing a large or bulky target antigen.
- Preferred embodiments include methods of treating cancer comprising administration of innate and adaptive immune cells, wherein said immune cells have been modified with a chimeric antigen receptor.
- Preferred methods include embodiments wherein said innate immune cells are macrophages and/or an ex vivo population of CD14 expressing cells.
- Preferred methods include embodiments wherein said macrophages are M1 macrophages.
- Preferred methods include embodiments wherein is macrophages are capable of producing more nitric oxide and less arginase upon activation through TLR4 as compared to control macrophages.
- Preferred methods include embodiments wherein said macrophages express CD16.
- Preferred methods include embodiments wherein said macrophages express CD25.
- Preferred methods include embodiments wherein said macrophages express CCR7.
- Preferred methods include embodiments wherein said macrophages express CD86
- Preferred methods include embodiments wherein said macrophages express CD127.
- Preferred methods include embodiments wherein said macrophages express interleukin-1 beta receptor.
- Preferred methods include embodiments wherein said macrophages express interleukin 10 receptor.
- Preferred methods include embodiments wherein said macrophages express TNF receptor p55.
- Preferred methods include embodiments wherein said macrophages express TNF receptor p75.
- Preferred methods include embodiments wherein said macrophages express CD215.
- Preferred methods include embodiments wherein said macrophages secrete IL-1 beta.
- Preferred methods include embodiments wherein said macrophages secrete IL-6.
- Preferred methods include embodiments wherein said macrophages secrete IL-8.
- Preferred methods include embodiments wherein said macrophages secrete IL-12.
- Preferred methods include embodiments wherein said macrophages secrete IL-15.
- Preferred methods include embodiments wherein said macrophages secrete IL-17.
- Preferred methods include embodiments wherein said macrophages are engineered to express HMGB-1.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-12.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-15.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-17.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-18.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-23.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-27.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-33.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-37.
- Preferred methods include embodiments wherein said macrophages are engineered to express a chimeric antigen receptor (CAR).
- CAR chimeric antigen receptor
- Preferred methods include embodiments wherein the CAR comprises (i) an extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain containing an intracellular signaling domain; and (b) a pharmaceutically acceptable carrier or excipient; thereby treating the cancer in the human subject.
- the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain, an Fc.epsilon.R intracellular signaling domain, an Fc.gamma.R intracellular signaling domain, or a TRIF intracellular signaling domain.
- Preferred methods include embodiments wherein the intracellular domain comprises two or more intracellular signaling domains.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR4 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR2 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR3 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR5 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR7/8 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR9 transmembrane domain.
- Preferred methods include embodiments wherein the extracellular domain further comprises a CD8a hinge domain.
- Preferred methods include embodiments wherein said macrophages are CD14+ cells comprises a population of CD14+/CD16+ cells.
- Preferred methods include embodiments wherein said macrophages are CD14+ cells comprises a population of CD14+/CD56+ cells.
- Preferred methods include embodiments wherein said macrophages comprise a population of CD14+ monocytes, a population of CD14+ macrophages or a population of CD14+ dendritic cells.
- Preferred methods include embodiments wherein said macrophages are CD14+ cells that are derived from the human subject.
- Preferred methods include embodiments wherein the population of CD14+ cells is obtained from a leukapheresis sample, a blood sample, or a PBMC sample from the human subject.
- Preferred methods include embodiments wherein the population of CD14+ cells is an ex vivo population of virally transduced cells.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells comprises a viral component.
- Preferred methods include embodiments wherein the antigen binding domain is a single domain antibody (sdAb) or a single chain variable fragment (scFv).
- sdAb single domain antibody
- scFv single chain variable fragment
- Preferred methods include embodiments wherein the antigen binding domain is an anti-HER2/neu binding domain.
- Preferred methods include embodiments wherein the sequence of the recombinant polynucleic acid encoding the CAR is from a viral vector.
- Preferred methods include embodiments wherein the method further comprises transducing a viral vector into a population of CD14+ cells ex vivo, thereby obtaining the ex vivo population of CD14+ cells comprising the recombinant polynucleic acid with a sequence encoding a CAR.
- Preferred methods include embodiments wherein the method comprises (i) extracting a blood sample from the human subject; (ii) isolating monocytes from the blood sample; and (iii) transfecting the monocytes from the blood sample with the recombinant polynucleic acid with a sequence encoding a CAR; and wherein administering comprises infusing.
- Preferred methods include embodiments wherein the recombinant polynucleic acid is mRNA.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells stimulates killing of cancer cells in the human subject by T cells of the human subject.
- Preferred methods include embodiments wherein the intracellular domain of the CAR is capable of inducing monocytic differentiation to M1 macrophages in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells enhances or improves effector function of a T cell in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells directly kills cancer cells in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells inhibits macrophage or macrophage related cells of the human subject from promoting tumor growth.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells is phagocytic.
- Preferred methods include embodiments wherein the cancer is a lymphoma.
- Preferred methods include embodiments wherein the cancer is a solid tumor.
- Preferred methods include embodiments wherein the cancer is a breast cancer.
- Preferred methods include embodiments wherein the cancer is a metastatic cancer.
- Preferred methods include embodiments wherein the cancer is an ErbB-2-expressing cancer.
- Preferred methods include embodiments wherein the method further comprises administering GM-CSF, IL-2, an agent that blocks CD47 activity or an agent that induces immunogenic cell death to the human subject.
- Preferred methods include embodiments wherein the method further comprises administering G-CSF.
- Preferred methods include embodiments wherein the method further comprises administering M-CSF.
- Preferred methods include embodiments wherein the method further comprises administering IL-2.
- Preferred methods include embodiments wherein the method further comprises administering antibody to interleukin-10.
- Preferred methods include embodiments wherein the method further comprises administering TNF-alpha.
- Preferred methods include embodiments wherein the method further comprises administering lymphotoxin.
- Preferred methods include embodiments wherein the method further comprises administering interferon alpha.
- Preferred methods include embodiments wherein the method further comprises administering interferon beta.
- Preferred methods include embodiments wherein the method further comprises administering interferon gamma.
- Preferred methods include embodiments wherein the method further comprises administering interferon tau.
- Preferred methods include embodiments wherein the method further comprises administering interferon omega.
- Preferred methods include embodiments wherein the method further comprises administering IL-7.
- Preferred methods include embodiments wherein the method further comprises administering IL-9.
- Preferred methods include embodiments wherein the method further comprises administering IL-12.
- Preferred methods include embodiments wherein the method further comprises administering IL-15.
- Preferred methods include embodiments wherein the method further comprises administering IL-18.
- Preferred methods include embodiments wherein the method further comprises administering IL-23.
- Preferred methods include embodiments wherein the method further comprises administering IL-33.
- Preferred methods include embodiments wherein the method further comprises administering C5 component of complement.
- Preferred methods include embodiments wherein the method further comprises administering C3 component of complement.
- Preferred methods include embodiments wherein the method further comprises administering BCG.
- Preferred methods include embodiments wherein the method further comprises administering a chemotherapeutic agent.
- Preferred methods include embodiments wherein the method further comprises administering an immunotherapeutic agent.
- Preferred methods include embodiments wherein the method further comprises administering an immunotherapeutic agent.
- Preferred methods include embodiments wherein the method further comprises administering radiotherapy.
- Preferred methods include embodiments wherein said innate immune cell is a natural killer (NK) cell.
- NK natural killer
- Preferred methods include embodiments wherein said NK cell is cytotoxic towards K562 cells.
- Preferred methods include embodiments wherein said NK cell expresses CD25.
- Preferred methods include embodiments wherein said NK cell expresses CD69.
- Preferred methods include embodiments wherein said NK cell expresses CD133.
- Preferred methods include embodiments wherein said NK cell expresses CD56.
- Preferred methods include embodiments wherein said NK cell expresses CD16.
- Preferred methods include embodiments wherein said NK cell expresses CD57.
- Preferred methods include embodiments wherein said NK cell expresses Fas ligand.
- Preferred methods include embodiments wherein said NK cell expresses IL-7 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-3 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-10 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-12 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-15 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-18 receptor.
- Preferred methods include embodiments wherein said NK cells are derived from peripheral blood.
- Preferred methods include embodiments wherein said NK cells are derived from umbilical cord blood.
- Preferred methods include embodiments wherein said NK cells are derived from menstrual blood.
- Preferred methods include embodiments wherein said NK cells are derived from bone marrow.
- Preferred methods include embodiments wherein said NK cells are derived from mobilized peripheral blood.
- Preferred methods include embodiments wherein mobilization of blood is achieved by treatment of the patient with agents that increase oxidative stress.
- Preferred methods include embodiments wherein said agents that increase oxidative stress are ozone.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with G-CSF.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with GM-CSF.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with M-CSF.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with mozibil.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with VEGF.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with thrombopoietin.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with TNF-alpha.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with hepatocyte growth factor.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with flt-3 ligand.
- Preferred methods include embodiments wherein said NK cells are generated from progenitor cells.
- Preferred methods include embodiments wherein said progenitors are umbilical cord derived.
- Preferred methods include embodiments wherein said progenitors are placental derived.
- Preferred methods include embodiments wherein said progenitors are CD34 derived.
- Preferred methods include embodiments wherein said progenitors are CD133 derived.
- Preferred methods include embodiments wherein said progenitors are inducible pluripotent stem cell derived.
- Preferred methods include embodiments wherein said progenitors are embryonic stem cell derived.
- Preferred methods include embodiments wherein said progenitors are parthenogenic stem cell derived.
- Preferred methods include embodiments wherein said progenitors are lymphoid progenitor cell derived.
- Preferred methods include embodiments wherein said NK cells are engineered to express a chimeric antigen receptor (CAR).
- CAR chimeric antigen receptor
- Preferred methods include embodiments wherein the CAR comprises (i) an extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain containing an intracellular signaling domain; and (b) a pharmaceutically acceptable carrier or excipient; thereby treating the cancer in the human subject.
- the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain, an Fc.epsilon.R intracellular signaling domain, an Fc.gamma.R intracellular signaling domain, or a TRIF intracellular signaling domain.
- Preferred methods include embodiments wherein the intracellular domain comprises two or more intracellular signaling domains.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR4 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR2 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR3 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR5 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR7/8 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR9 transmembrane domain.
- Preferred methods include embodiments wherein the extracellular domain further comprises a CD8a hinge domain.
- Preferred methods include embodiments wherein said NK are pre-activated by culture in IL-2.
- Preferred methods include embodiments wherein said NK cells are pre-activated by culture with dendritic cells.
- Preferred methods include embodiments wherein said NK cells are pre-activated by culture with monocytes.
- Preferred methods include embodiments wherein said NK cells are capable of proliferating approximately one multiplication ever 15-36 hours.
- Preferred methods include embodiments wherein said NK cells are capable of proliferating approximately one multiplication ever 20-30 hours.
- Preferred methods include embodiments wherein said NK cells are capable of proliferating approximately one multiplication ever 15-36 hours.
- Preferred methods include embodiments wherein NK cells are virally transfected.
- Preferred methods include embodiments wherein the antigen binding domain is a single domain antibody (sdAb) or a single chain variable fragment (scFv).
- sdAb single domain antibody
- scFv single chain variable fragment
- Preferred methods include embodiments wherein the antigen binding domain is an anti-HER2/neu binding domain.
- Preferred methods include embodiments wherein the sequence of the recombinant polynucleic acid encoding the CAR is from a viral vector.
- Preferred methods include embodiments wherein the method further comprises transducing a viral vector into a population of CD56+ cells ex vivo, thereby obtaining the ex vivo population of CD56+ cells comprising the recombinant polynucleic acid with a sequence encoding a CAR.
- Preferred methods include embodiments wherein the method comprises (i) extracting a blood sample from the human subject; (ii) isolating NK cells from the blood sample; and (iii) transfecting the NK cells from the blood sample with the recombinant polynucleic acid with a sequence encoding a CAR; and wherein administering comprises infusing.
- Preferred methods include embodiments wherein the recombinant polynucleic acid is mRNA.
- Preferred methods include embodiments wherein the ex vivo population of CD56+ cells stimulates killing of cancer cells in the human subject by T cells of the human subject.
- Preferred methods include embodiments wherein the intracellular domain of the CAR is capable of inducing activation of NK cell in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD56+ cell enhances or improves effector function of a T cell in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD56+ cells directly kills cancer cells in the human subject.
- Preferred methods include embodiments wherein adaptive immune cell is a T cell.
- Preferred methods include embodiments wherein said T cell is a CD4 T cell.
- Preferred methods include embodiments wherein said T cell is a CD8 T cell.
- Preferred methods include embodiments wherein said T cell is an NKT cell.
- Preferred methods include embodiments wherein said T cell is a double negative T cell.
- Preferred methods include embodiments wherein said T cell expresses interleukin-2 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-4 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-7 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-9 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-12 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-15 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-17 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-18 receptor.
- Preferred methods include embodiments wherein said NK cells are engineered to express a chimeric antigen receptor (CAR).
- CAR chimeric antigen receptor
- Preferred methods include embodiments wherein the CAR comprises (i) an extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain containing an intracellular signaling domain; and (b) a pharmaceutically acceptable carrier or excipient; thereby treating the cancer in the human subject.
- the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain, an Fc.epsilon.R intracellular signaling domain, an Fc.gamma.R intracellular signaling domain, or a TRIF intracellular signaling domain.
- Preferred methods include embodiments wherein the intracellular domain comprises two or more intracellular signaling domains.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR4 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR2 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR3 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR5 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR7/8 transmembrane domain.
- transmembrane domain comprises a CD8a transmembrane domain or a TLR9 transmembrane domain.
- Preferred methods include embodiments wherein the extracellular domain further comprises a CD8a hinge domain.
- Preferred methods include embodiments wherein said T cells are pre-activated by culture in IL-2.
- Preferred methods include embodiments wherein said T cells are pre-activated by culture with dendritic cells.
- Preferred methods include embodiments wherein said T cells are pre-activated by culture with monocytes.
- Preferred methods include embodiments wherein said T cells are capable of proliferating approximately one multiplication ever 15-36 hours.
- Preferred methods include embodiments wherein said T cells are capable of proliferating approximately one multiplication ever 20-30 hours.
- Preferred methods include embodiments wherein said T cells are capable of proliferating approximately one multiplication ever 15-36 hours.
- Preferred methods include embodiments wherein T cells are virally transfected.
- Preferred methods include embodiments wherein the antigen binding domain is a single domain antibody (sdAb) or a single chain variable fragment (scFv).
- sdAb single domain antibody
- scFv single chain variable fragment
- Preferred methods include embodiments wherein the antigen binding domain is an anti-HER2/neu binding domain.
- Preferred methods include embodiments wherein the sequence of the recombinant polynucleic acid encoding the CAR is from a viral vector.
- Preferred methods include embodiments wherein CAR-macrophages are injected initially, alone or in combination with CAR-NK.
- Preferred methods include embodiments wherein CAR-T cells are administered at a timepoint after which CAR-macrophages and/or CAR-NK have modified to tumor microenvironment to allow for CAR-T cells to enter said tumor microenvironment.
- Preferred methods include embodiments wherein all cells expressing CAR possess an inhibitor of NR2F6.
- the invention provides means of treating cancer through inactivating the tumor microenvironment followed by administration of CAR-T cells.
- the invention provides the use of CAR-Macrophages and CAR-NK cells for manipulation of the tumor microenvironment in order to decrease immunesuppressinvess and thus allowing for entry of CAR-T cells into solid tumors.
- the role of the CAR-macrophages and CAR-NK cells are to increase propensity for Th1 immunity and allow for activity of CAR-T cells which otherwise is blunted by mediators such as PGE-2, IL-10, TGF-beta and soluble TNF-alpha receptor found in the tumor microenvironment.
- an element means one element or more than one element.
- compositions, and methods are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions, methods, and devices can also “consist essentially of” or “consist of” the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.
- Activation refers to the state of a MIL that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be as sociated with induced cytokine production, and detectable effector functions.
- activated MILs refers to, among other things, MILs that are undergoing cell division.
- antibody refers to an immunoglobulin molecule which specifically binds with an antigen.
- Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
- the antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.
- antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
- antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.
- antigen as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
- any macromolecule including virtually all proteins or peptides, can serve as an antigen.
- antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
- an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the embodiments include, but are not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
- anti-tumor effect refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
- An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies to prevent the occurrence of tumor in the first place.
- auto-antigen means any self-antigen which is mistakenly recognized by the immune system as being foreign.
- Auto-antigens comprise, but are not limited to, cellular proteins, phosphoproteins, cellular surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.
- autologous is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
- Allogeneic refers to a graft derived from a different animal of the same species.
- Xenogeneic refers to a graft derived from an animal of a different species.
- cancer as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may include non-solid tumors (such as hematological tumors, for example, myeloma, leukemias and lymphomas) or may include solid tumors.
- non-solid tumors such as hematological tumors, for example, myeloma, leukemias and lymphomas
- Types of cancers to be treated with the CARs as described herein include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
- carcinoma a malignant neoplasm
- blastoma a malignant neoid tumors
- malignancies e.g., sarcomas, carcinomas, and melanomas.
- adult tumors/cancers and pediatric tumors/cancers are also included.
- Co-stimulatory ligand includes a molecule on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a MIL, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a MIL response, including, but not limited to, proliferation, activation, differentiation, and the like.
- an antigen presenting cell e.g., an aAPC, dendritic cell, B cell, and the like
- a co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.
- a co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a MIL, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
- a co-stimulatory molecule present on a MIL such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
- a “co-stimulatory molecule” refers to the cognate binding partner on a MIL that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the MIL, such as, but not limited to, proliferation.
- Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.
- a “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to MIL proliferation and/or upregulation or downregulation of key molecules.
- a “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
- a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
- an “effective amount” as used herein means an amount which provides a therapeutic or prophylactic benefit.
- Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
- a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
- Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
- endogenous refers to any material from or produced inside an organism, cell, tissue or system.
- exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
- expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
- “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
- An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
- Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
- “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
- the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared .times.100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
- the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
- immunoglobulin or “Ig,” as used herein is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE.
- isolated means altered or removed from the natural state.
- a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
- An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
- A refers to adenosine
- C refers to cytosine
- G refers to guanosine
- T refers to thymidine
- U refers to uridine.
- a “Lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
- nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
- operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
- a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
- operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
- tumor antigen or “overexpression” of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ.
- Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.
- parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
- patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
- the patient, subject or individual is a human.
- peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
- a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
- Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
- the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
- Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
- the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
- promoter as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
- a classical chimeric antigen receptor is a chimeric type I trans-membrane protein which connects an extracellular antigen-recognizing domain (binder) to an intracellular signaling domain (endodomain).
- the binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site.
- scFv single-chain variable fragment
- mAb monoclonal antibody
- a spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation.
- a common spacer domain used is the Fc of IgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the IgG1 hinge alone, depending on the antigen.
- a trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.
- Early CAR designs had endodomains derived from the intracellular parts of either the .gamma. chain of the Fc.epsilon.R1 or CD3.zeta. Consequently, these first generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive.
- compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3.zeta. results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition.
- the co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal—namely immunological signal 2, which triggers T-cell proliferation.
- Some receptors have also been described which include TNF receptor family endodomains, such as the closely related OX40 and 41 BB which transmit survival signals.
- Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards tumor cells expressing the targeted antigen.
- CARs typically therefore comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain which comprises or associates with a signaling domain.
- a CAR may have the general structure: Antigen binding domain-spacer domain-transmembrane domain-intracellular signaling domain (endodomain).
- CARs may be utilized for application in innate cells, such as NK and macrophages, as well as in their more classical embodiment, T cells.
- the invention provides means of inducing an anti-cancer response in a mammal, comprising the steps of initially “priming” the mammal by administering an agent that causes local accumulation of CAR-macrophage. Subsequently, a tumor antigen is administered in the local area where said agents causing accumulation of antigen presenting cells is administered. A time period is allowed to pass to allow for said antigen presenting cells to traffic to the lymph nodes. Subsequently a maturation signal, or a plurality of maturation signals are administered to enhance the ability of said antigen presenting cell to activate adaptive immunity. In some embodiments of the invention activators of adaptive immunity are concurrently given, as well as inhibitors of the tumor derived inhibitors are administered to derepress the immune system.
- priming of the patient is achieved by administration of GM-CSF subcutaneously in the area in which antigen is to be injected.
- GM-CSF GM-CSF
- Various scenarios are known in the art for administration of GM-CSF prior to administration, or concurrently with administration of antigen.
- the practitioner of the invention is referred to the following publications for dosage regimens of GM-CSF and also of peptide antigens.
- the invention calls for administration of tumor antigen.
- Various tumor antigens may be utilized, in one preferred embodiment, lysed tumor cells from the same patient area utilized. Means for generation of lysed tumor cells are well known in the art and described in the following references.
- One example method for generation of tumor lysate involves obtaining frozen autologous samples which are placed in hanks buffered saline solution (HBSS) and gentamycin 50.mu.g/ml followed by homogenization by a glass homogenizer. After repeated freezing and thawing, particle-containing samples are selected and frozen in aliquots after radiation with 25 kGy. Quality assessment for sterility and endotoxin content is performed before freezing. Cell lysates are subsequently administered into the patient in a preferred manner subcutaneously at the local areas where DC priming was initiated. After 12-72 hours, the patient is subsequently administered with an agent capable of inducing maturation of DC.
- HBSS hanks buffered saline solution
- gentamycin 50.mu.g/ml gentamycin 50.mu.g/ml
- particle-containing samples are selected and frozen in aliquots after radiation with 25 kGy. Quality assessment for sterility and endotoxin content is performed
- Agents useful for the practice of the invention include BCG and HMGB1 peptide.
- Other useful agents include: a) histone DNA; b) imiqimod; c) beta-glucan; d) hsp65; e) hsp90; 0 HMGB-1; g) lipopolysaccharide; h) Pam3CSK4; i) Poly I: Poly C; j) Flagellin; k) MALP-2; 1) Imidazoquinoline; m) Resiquimod; n) CpG oligonucleotides; o) zymosan; p) peptidoglycan; q) lipoteichoic acid; r) lipoprotein from gram-positive bacteria; s) lipoarabinomannan from mycobacteria; t) Polyadenylic-polyuridylic acid; u) monophosphoryl lipid A; v) single stranded RNA; w) double
- siRNA or shRNA containing the effector sequences a) UUAUAAUGACUGGAUGUUC; b) GUCUGGUGUAUGAAGGGUU; c) CUCCUAUUUUGGUUUAUGC and d) GCAGCGUCUUUCAGUGCUU.
- siRNA or shRNA may be administered through various modalities including biodegradable matrices, pressure gradients or viral transfect.
- autologous dendritic cells are generated and IDO is silenced, prior to, concurrent with or subsequent to silencing, said dendritic cells are pulsed with tumor antigen and administered systemically.
- mature DC are modified with CAR transfection prior to administration.
- Culture of dendritic cells is well known in the art, for example, U.S. Pat. No. 6,936,468, issued to Robbins, et al., for the use of tolerogenic dendritic cells for enhancing tolerogenicity in a host and methods for making the same.
- the current invention aims to reduce tolerogenesis, the essential means of dendritic cell generation are disclosed in the patent.
- U.S. Pat. No. 6,734,014 issued to Hwu, et al., for methods and compositions for transforming dendritic cells and activating T cells.
- recombinant dendritic cells are made by transforming a stem cell and differentiating the stem cell into a dendritic cell.
- the resulting dendritic cell is said to be an antigen presenting cell which activates T cells against MHC class I-antigen targets.
- Antigens for use in dendritic cell loading are taught in, e.g., U.S. Pat. No. 6,602,709, issued to Albert, et al. This patent teaches methods for use of apoptotic cells to deliver antigen to dendritic cells for induction or tolerization of T cells.
- the methods and compositions are said to be useful for delivering antigens to dendritic cells that are useful for inducing antigen-specific cytotoxic T lymphocytes and T helper cells.
- the disclosure includes assays for evaluating the activity of cytotoxic T lymphocytes.
- the antigens targeted to dendritic cells are apoptotic cells that may also be modified to express non-native antigens for presentation to the dendritic cells.
- the dendritic cells are said to be primed by the apoptotic cells (and fragments thereof) capable of processing and presenting the processed antigen and inducing cytotoxic T lymphocyte activity or may also be used in vaccine therapies.
- 6,455,299 issued to Steinman, et al., teaches methods of use for viral vectors to deliver antigen to dendritic cells. Methods and compositions are said to be useful for delivering antigens to dendritic cells, which are then useful for inducing T antigen specific cytotoxic T lymphocytes.
- the disclosure provides assays for evaluating the activity of cytotoxic T lymphocytes.
- Antigens are provided to dendritic cells using a viral vector such as influenza virus that may be modified to express non-native antigens for presentation to the dendritic cells.
- the dendritic cells are infected with the vector and are said to be capable of presenting the antigen and inducing cytotoxic T lymphocyte activity or may also be used as vaccines.
- Immune cells for use in the practice of the invention include DCs, the presence of which may be checked in the previously described method, are preferably selected from myeloid cells (such as monocytic cells and macrophages) expressing langerin, MHC (major histocompatibility complex) class II, CCR2 (chemokine (C-C motif) receptor 2), CX3CR1 and/or Grl molecules in mice; myeloid cells expressing CD14, CD16, HLA dR (human leukocyte antigen disease resistance) molecule, langerin, CCR2 and/or CX3CR1 in humans; dendritic cells expressing CD11c, MHC class II molecules, and/or CCR7 molecules; and IL-1.beta. producing dendritic cells.
- myeloid cells such as monocytic cells and macrophages
- myeloid cells such as monocytic cells and macrophages
- myeloid cells such as monocytic cells and macrophages
- MHC major histocompatibility complex
- CD8 T cells are preferably selected from CD3+, CD4+ and/or CD8+T lymphocytes, FOXP3 (forkhead box P3) T lymphocytes, Granzyme B/TIA (Tcell-restricted intracellular antigen) T lymphocytes, and Tc1 cells (IFN-.gamma. producing CD8+T lymphocytes).
- Immune cells expressing a protein that binds calreticulin may be selected from cells expressing at least one of the following proteins: LRP1 (Low density lipoprotein receptor-related protein 1, CD91), Ca.sup.++-binding proteins such as SCARF1 and SCARF2, MSR1 (Macrophage scavenger receptor 1), SRA, CD59 (protectin), CD207 (langerin), and THSDI (thrombospondin).
- LRP1 Low density lipoprotein receptor-related protein 1, CD91
- Ca.sup.++-binding proteins such as SCARF1 and SCARF2, MSR1 (Macrophage scavenger receptor 1), SRA, CD59 (protectin), CD207 (langerin), and THSDI (thrombospondin).
- Macrophages selectively phagocytose tumor cells, but this process is countered by protective molecules on tumor cells such as CD47, which binds macrophage signal-regulatory protein a to inhibit phagocytosis.
- Blockade of CD47 on tumor cells leads to phagocytosis by macrophages.
- CAR-MSC are administered together with an agent that blocks CD47 activity. It has been demonstrated that activation of TLR signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance tumor phagocytosis.
- Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages.
- Calreticulin previously shown to be a protein found on cancer cells that activated macrophage phagocytosis of tumors, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.
- TLR agonists are administered that stimulate expression of calreticulin and/or enhance macrophage phagocytosis of tumors.
- IL-27 induces macrophage ability to kill tumor cells in vitro and in vivo, as well as altering the tumor promoting M2/myeloid suppressor cells into tumoricidal cells.
- addition of IL-27 or compounds capable of activating the IL-27 receptor signaling are administered together with IL-27 to enhance tumor phagocytosis by macrophages.
- Tumor-associated macrophages deriving from monocytes or migrating into the tumor, are an important constituent of tumor microenvironments, which in many cases modulates tumor growth, tumor angiogenesis, immune suppression, metastasis and chemoresistance.
- Mechanisms of macrophage promotion of tumor growth include production of EGF, M-CSF, and VEGF.
- Macrophage infiltration of tumors is associated with poor prognosis in renal, melanoma, breast, pancreatic, lung, endometrial, bladder, prostate. Tumor growth are inhibited when monocytes/macrophages are ablated.
- CAR-macrophages are utilized to force the tumor microenvironment to stimulate tumor killing and inhibit macrophage or macrophage related cells from promoting tumor growth.
- drugs targeting tumor-associated macrophages especially c-Fms kinase inhibitors and humanized antibodies targeting colony-stimulating factor-1 receptor, are envisioned.
- Tumors mediate various effects to reprogram macrophages, these are usually mediated via IL-10 and other cytokines such as VEGF, TGF-beta, and M-CSF, which cause macrophages to lose tumor cytotoxicity and shift into tumor promoting, immune suppressive, angiogenic supporting cells.
- cytokines such as VEGF, TGF-beta, and M-CSF
- myeloid derived suppressor cells are similar to myeloid progenitor cells, or the previously described “natural suppressor” cell.
- Irradiated tissues induce a TLR-1 reprogramming of macrophages to promote tumor growth and angiogenesis.
- Macrophage promotion of tumor growth is seen in numerous situations, in one example, treating of tumor bearing animals with BRAF inhibitors results in upregulation of macrophage production of VEGF which accelerates tumor growth.
- tumors produce factors such as GM-CSF which in part stimulate macrophages to produce CCL18, which promotes tumor metastasis.
- the lactic acid microenvironment of the tumor has been shown to promote skewing of macrophages towards at tumor-promoting M2 type.
- lactic acid produced by tumor cells as a by-product of aerobic or anaerobic glycolysis, possesses an essential role in inducing the expression of VEGF and the M2-like polarization of tumor-associated macrophages, specifically inducing expression of arginase 1 through a HIF-1alpha dependent pathway.
- LDH-A lactate dehydrogenase-A
- siRNA silencing of LDH-A in Pan02 pancreatic cancer cells that are injected in C57BL/6 mice results in development of smaller tumors than mice injected with wild type, non-silenced Pan02 cells.
- NK cells from LDH-A-depleted tumors had improved cytolytic function.
- Exogenous lactate administration was shown to increase the frequency of MDSCs generated from mouse bone marrow cells with GM-CSF and IL-6 in vitro.
- lactate pretreatment of NK cells in vitro inhibited cytolytic function of both human and mouse NK cells. This reduction of NK cytotoxic activity was accompanied by lower expression of perforin and granzyme in NK cells. The expression of NKp46 was lower in lactate-treated NK cells.
- depletion of glucose levels using a ketogenic diet to lower lactate production by glycolytic tumors is utilized to augment therapeutic effects of CAR-macrophage.
- Utilization of ketogenic diet has been previously described for immune modulation, and cancer therapy.
- Specific quantification of intratumoral lactate and its manipulation has been described and incorporated by reference.
- Potentiation of chemotherapeutic and radiotherapeutic effects by ketogenic diets have been reported and techniques are incorporated by reference for use with the current CAR-macrophage invention.
- ketogenic diet Suppression of tumor growth and activity induced by ketogenic diet may be augmented by addition of hyperbaric oxygen, thus in one embodiment of the invention, the utilization of oxidative therapies, as disclosed in references incorporated, together with ketogenic diet is utilized to augment therapeutic efficacy of CAR-macrophage.
- Dopamine possesses antiangiogenic effects as well as myeloprotective effects, in one embodiment of the invention addition of dopamine to the CAR-macrophage treatment is disclosed.
- Vinblastine is a widely used chemotherapeutic agent that has been demonstrated to induce dendritic cell maturation.
- CAR-macrophage are utilized together with vinblastine therapy to induce augmented anticancer activity.
- Oxiplatin and anthracyclines have been demonstrated to not only directly kill tumor cells but also stimulate T cell immunity against tumor cells. It was demonstrated that these agents induce a rapid and prominent invasion of interleukin (IL)-17-producing .gamma..delta.
- IL interleukin
- V.gamma.4(+) and V.gamma.6(+) T lymphocytes (.gamma.6 T17 cells) that precedes the accumulation of CD8 CTLs within the tumor bed.
- T cell receptor .delta.( ⁇ / ⁇ ) or V.gamma.4/6( ⁇ / ⁇ ) mice the therapeutic efficacy of chemotherapy was reduced and furthermore no IL-17 was produced by tumor-infiltrating T cells, and CD8 CTLs did not invade the tumor after treatment.
- .gamma..delta the therapeutic efficacy of chemotherapy was reduced and furthermore no IL-17 was produced by tumor-infiltrating T cells, and CD8 CTLs did not invade the tumor after treatment.
- Th17 cells could produce both IL-17A and IL-22, the absence of a functional IL-17A-IL-17R pathway significantly reduced tumor-specific T cell responses elicited by tumor cell death, and the efficacy of chemotherapy in four independent transplantable tumor models.
- the adoptive transfer of .gamma..delta. T cells to naive mice restored the efficacy of chemotherapy in IL-17A( ⁇ / ⁇ ) hosts.
- the anticancer effect of infused .gamma..delta. T cells was lost when they lacked either IL-1R1 or IL-17A.
- Intratumoral injection of dendritic cells stimulates antitumor immunity in vivo in clinical situations, suggesting that modulating the antigen presenting cell in the tumor microenvironment will induce an antitumor response.
- Administration of radiotherapy to tumors to induce immunogenic cell death, followed by intratumoral administration of DC has been demonstrated to result in enhanced antigen presentation, accordingly, this technique may be modified to enhance effects of CAR-macrophages.
- the induction of immunity to tumors in the present invention is associated with the unique nature of: a) ongoing basal cell death within the tumor; and b) cell death induced by chemotherapy, radiotherapy, hyperthermia, or otherwise induced cell death.
- Cell death can be classified according to the morphological appearance of the lethal process (that may be apoptotic, necrotic, autophagic or associated with mitosis), enzymological criteria (with and without the involvement of nucleases or distinct classes of proteases, like caspases), functional aspects (programmed or accidental, physiological or pathological) or immunological characteristics (immunogenic or non-immunogenic).
- Cell death is defined as “immunogenic” or “immune stimulatory” if dying cells that express a specific antigen (for example a tumor associated antigen, phosphotidyl serine, or calreticulin), yet are uninfected (and hence lack pathogen-associated molecular patterns), and are injected subcutaneously into mice, in the absence of any adjuvant, cause a protective immune response against said specific antigen.
- a protective immune response precludes the growth of living transformed cells expressing the specific antigen injected into mice.
- cancer cells succumb to an immunogenic cell death (or immunogenic apoptosis) modality, they stimulate the immune system, which then mounts a therapeutic anti-cancer immune response and contributes to the eradication of residual tumor cells.
- cancer cells succumb to a non-immunogenic death modality, they fail to elicit such a protective immune response.
- the tumor derived immune suppressive molecules contribute to general inhibition or inability of the tumor to be eliminated.
- CAR-macrophage are administered concurrently, prior to, or subsequent to administration of an agent that induces immunogenic cell death in a patient. Methods of determining whether compounds induce immunogenic cell death are known in the art and include the following, which was described by Zitvogel et al.
- step (a) treating the cells, the mammalian cells and inducing the cell death or apoptosis, typically of mammalian cancer cells capable of expressing calreticulin (CRT), by exposing said mammalian cells to a particular drug (the test drug), for example 18 hours;
- step (b) inoculating (for example intradermally) the dying mammalian cells from step (a) in a particular area (for example a flank) of the mammal, typically a mouse, to induce an immune response in this area of the mammal;
- step (d) comparing the size of the tumor in the inoculated mammal with a control mammal also exposed to the minimal tumorigenic dose of syngeneic live tumor cells of step (c) [for example a
- in vitro means are available for assessing the ability of various drugs or therapeutic approaches to induce immunogenic cell death.
- Specific characteristics to assess when screening for immunogenic cell death include: a) ability to induce dendritic cell maturation in vitro; b) ability to activate NK cells; and c) ability to induce activation of gamma delta T cells or NKT cells.
- Specific drugs known to induce immunogenic cell death include oxiplatine and anthracyclines, as well as radiotherapy, and hyperthermia.
- certain chemotherapies that activate TLR4 through induction of HMGB1 have been observed to function suboptimally in patients that have a TLR4 polymorphism, thus suggesting actual contribution of TLR activation as a means of chemotherapy inhibition of cancer.
- oncoviruses or oncolytic viruses are known to induce immunogenic cell death and may be useful for the practice of the invention.
- the CAR-macrophage disclosed in the invention may be utilized in combination with conventional immune modulators including BCG, CpG DNA, interferon alpha, tumor bacterial therapy, checkpoint inhibitors, Treg depleting agents, and low dose cyclophosphamide.
- CAR-macrophage cells are generated with specificity towards ROBO-4.
- Numerous means of generating CAR-T cells are known in the art, which are applied to CAR-macrophage.
- FMC63-28z CAR (Genebank identifier HM852952.1), is used as the template for the CAR except the anti-CD19, single-chain variable fragment sequence is replaced with an ROBO-4 fragment.
- the construct is synthesized and inserted into a pLNCX retroviral vector.
- Retroviruses encoding the ROBO-4-specific CAR are generated using the retrovirus packaging kit, Ampho (Takara), following the manufacturer's protocol.
- PBMCs are plated at 2.times.10(6) cells/mL in cell culture for 2 hours and the adherent cells are collected. The cells were then stimulated for 2 days on a tissue-culture-treated 24-well plate containing M-CSF at a concentration of 100 ng/ml
- a 24-well plate are coated with RetroNectin (Takara) at 4.degree. C. overnight, according to the manufacturer's protocol, and then blocked with 2% BSA at room temperature for 30 min.
- the plate was then loaded with retrovirus supernatants at 300.mu.L/well and incubated at 37.degree. C. for 6 h.
- 1.times.10(6) stimulated adherent cells in 1 mL of medium are added to 1 mL of retrovirus supernatants before being transferred to the pre-coated wells and cultured at 37.degree. C. for 2 d.
- the cells are then transferred to a tissue-culture-treated plate at 1.times.10 (6) cells/mL and cultured in the presence of 100 U/mL of recombinant human M-CSF, applying the T cell protocol but not utilizing IL-2 or antiCD3/antiCD28.
- CARs Other means of generating CARs are known in the art and incorporated by reference. For example, Groner's group genetically modified T lymphocytes and endowed them with the ability to specifically recognize cancer cells. Tumor cells overexpressing the ErbB-2 receptor served as a model. The target cell recognition specificity was conferred to T lymphocytes by transduction of a chimeric gene encoding the zeta-chain of the TCR and a single chain antibody (scFv(FRP5)) directed against the human ErbB-2 receptor. The chimeric scFv(FRP5)-zeta gene was introduced into primary mouse T lymphocytes via retroviral gene transfer. Naive T lymphocytes were activated and infected by cocultivation with a retrovirus-producing packaging cell line.
- scFv(FRP5) single chain antibody
- the scFv(FRP5)-zeta fusion gene was expressed in >75% of the T cells. These T cells lysed ErbB-2-expressing target cells in vitro with high specificity.
- mice were treated with autologous, transduced T cells.
- the adoptively transferred scFv(FRP5)-zeta-expressing T cells caused total regression of ErbB-2-expressing tumors.
- the presence of the transduced T lymphocytes in the tumor tissue was monitored. No humoral response directed against the transduced T cells was observed. Abs directed against the ErbB-2 receptor were detected upon tumor lysis. Hornbach et al.
- an anti-CEA chimeric receptor whose extracellular moiety is composed of a humanized scFv derived from the anti-CEA mAb BW431/26 and the CH2/CH3 constant domains of human IgG.
- the intracellular moiety consists of the gamma-signaling chain of the human Fc epsilon RI receptor constituting a completely humanized chimeric receptor.
- the humBW431/26 scFv-CH2CH3-gamma receptor is expressed as a homodimer on the surface of MD45 T cells. Co-incubation with CEA+ tumor cells specifically activates grafted MD45 T cells indicated by IL-2 secretion and cytolytic activity against CEA+ tumor cells.
- the efficacy of receptor-mediated activation is not affected by soluble CEA up to 25 micrograms/ml demonstrating the usefulness of this chimeric receptor for specific cellular activation by membrane-bound CEA even in the presence of high concentrations of CEA, as found in patients during progression of the disease (200).
- CAR T cells Targeting of mucins associated with cancers has been performed with CAR T cells by grafting the antibody that binds to the mucin with CD3 zeta chain.
- this procedure is modified for CAR-macrophage.
- chimeric immune receptor consisting of an extracellular antigen-binding domain derived from the CC49 humanized single-chain antibody, linked to the CD3zeta signaling domain of the T cell receptor, was generated (CC49-zeta). This receptor binds to TAG-72, a mucin antigen expressed by most human adenocarcinomas.
- CC49-zeta was expressed in CD4+ and CD8+ T cells and induced cytokine production on stimulation.
- CC49-zeta Human T cells expressing CC49-zeta recognized and killed tumor cell lines and primary tumor cells expressing TAG-72. CC49-zeta T cells did not mediate bystander killing of TAG-72-negative cells. In addition, CC49-zeta T cells not only killed FasL-positive tumor cells in vitro and in vivo, but also survived in their presence, and were immunoprotective in intraperitoneal and subcutaneous murine tumor xenograft models with TAG-72-positive human tumor cells. Finally, receptor-positive T cells were still effective in killing TAG-72-positive targets in the presence of physiological levels of soluble TAG-72, and did not induce killing of TAG-72-negative cells under the same conditions.
- CAR T cells have been utilized targeting surface tumor antigens
- the main issue with this approach is the difficulty of T cells to enter tumors due to features specific to the tumor microenvironment. These include higher interstitial pressure inside the tumor compared to the surroundings, acidosis inside the tumor, and expression in the tumor of FasL which kills activated T cells.
- the invention seeks to more effectively utilize CAR-macrophage cells by directly targeting them to tumor endothelium, which is in direct contact with blood and therefore not susceptible to intratumoral factors the limit efficacy of conventional T cell therapies.
- CAR-macrophage are targeting to tumor antigens.
- protocols similar to Kershaw et al. are utilized with the exception that tumor endothelial antigens are targeted as opposed to conventional tumor antigens.
- tumor endothelial antigens include CD93, TEM-1, VEGFR1, and survivin.
- Antibodies can be made for these proteins, methodologies for which are described in U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, and 5,639,641.
- T cells with reactivity against the ovarian cancer-associated antigen alpha-folate receptor were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor gamma chain.
- Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1 received a dose escalation of T cells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specific T cells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells.
- PBMCs are derived from leukapheresis and CD14 monocytes are collected by MACS. After 3 days of culture, M-CSF at 100 ng/ml plasmid encoding the chimeric CAR-macrophage recognizing tumor specific antigen and subsequently selected for gene integration by culture in G418.
- M-CSF 100 ng/ml plasmid encoding the chimeric CAR-macrophage recognizing tumor specific antigen and subsequently selected for gene integration by culture in G418.
- stimulation of allogeneic monocytic cells is achieved by coculture of patient PBMCs with irradiated (5,000 cGy) allogeneic donor PBMCs from cryopreserved apheresis product (mixed lymphocyte reaction).
- the MHC haplotype of allogeneic donors is determined before use, and donors that differed in at least four MHC class I alleles from the patient are used.
- Culture medium consisted of AimV medium (Invitrogen, Carlsbad, Calif.) supplemented with 5% human AB.sup.-serum (Valley Biomedical, Winchester, Va.), penicillin (50 units/mL), streptomycin (50 mg/mL; Bio Whittaker, Walkersville, Md.), amphotericin B (Fungizone, 1.25 mg/mL; Biofluids, Rockville, Md.), L-glutamine (2 mmol/L; Mediatech, Herndon, Va.), and human recombinant IL-2 (Proleukin, 300 IU/mL; Chiron).
- PBMCs Mixed lymphocyte reaction consisted of 2.times.10.sup.6 patient monocytes and 1.times.10.sup.7 allogeneic stimulator PBMCs in 2 mL AimV per well in 24-well plates. Between 24 and 48 wells are cultured per patient for 3 days, at which time transduction is done by aspirating 1.5 mL of medium and replacing with 2.0 mL retroviral supernatant containing 300 IU/mL IL-2, 10 mmol/L HEPES, and 8.mu.g/mL polybrene (Sigma, St. Louis, Mo.) followed by covering with plastic wrap and centrifugation at 1,000.times.g for 1 hour at room temperature. After overnight culture at 37.degree.
- Restimulation is done by incubating patient T cells (1.times.10.sup.6/mL) and stimulator PBMCs (2.times.10.sup.6/mL) in 3-liter Fenwall culture bags in AimV+additives and IL-2 (no G418). Cell numbers were adjusted to 1.times.10.sup.6/mL, and IL-2 was added every 2 days, until sufficient numbers for treatment were achieved.
- the present invention relates to a strategy of adoptive cell transfer of monocytes or DC transduced to express a chimeric antigen receptor (CAR).
- CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., tumor endothelial antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor endothelium cellular immune activity.
- the present invention relates generally to the use of monocytes or DC cells genetically modified to stably express a desired CAR that possesses high affinity towards tumor associated endothelium.
- Monocytes or DC cells expressing a CAR are referred to herein as CAR-MACROPHAGE cells or CAR modified DC cells.
- the cell can be genetically modified to stably express an antibody binding domain on its surface, conferring novel antigen specificity that is MHC independent.
- the monocyte or DC cell is genetically modified to stably express a CAR that combines an antigen recognition domain of a specific antibody with an intracellular domain of the CD3-zeta chain or Fc.gamma.RI protein into a single chimeric protein.
- TLR signaling molecules are engineered in the intracellular portion of the CAR, said molecules include TRIF, TRADD, and MyD99.
- the CAR of the invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain.
- the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
- the transmembrane domain can be selected or modified by amino add substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
- the transmembrane domain is the CD8a hinge domain.
- the CAR of the invention can be designed to comprise the CD80 and/or CD86 and/or CD40L and/or OX40L signaling domain by itself or be combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention.
- the cytoplasmic domain of the CAR can be designed to further comprise the signaling domain of MyD88.
- the cytoplasmic domain of the CAR can include but is not limited to CD80 and/or CD86 and/or CD40L and/or OX40L signaling modules and combinations thereof.
- inhibition of TGF-beta is performed either by transfection with an shRNA possessing selectively towards TGF-beta or by constructing the CAR to possess a dominant negative mutant of TGF-beta receptor. This would render the CAR-macrophage cell resistant to inhibitory activities of the tumors. Accordingly, the invention provides CAR-macrophage cells and methods of their use for adoptive therapy.
- the CAR-macrophage cells of the invention can be generated by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-CD19, CD8a hinge and transmembrane domain, and MyD88, into the cells.
- a desired CAR for example a CAR comprising anti-CD19, CD8a hinge and transmembrane domain, and MyD88.
- the CAR-macrophage cells of the invention are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
- the chimeric antigen receptor may include two intracellular signaling domains.
- the chimeric antigen receptor may include a first intracellular signaling domain linked to the transmembrane domain and a second intracellular signaling domain linked to a terminal of the first intracellular signaling domain that is not linked with the transmembrane domain.
- the first intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 (CD134), OX40 ligand (OX40L, CD252), 4-1BB (CD137), CD28, DAP10, CD3-zeta (CD3) and DAP12
- the second intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12.
- at least one of the first intracellular signaling domain and the second intracellular signaling domain includes the whole or a portion of OX40 ligand.
- the chimeric antigen receptor may include a first intracellular signaling domain containing the whole or a portion of OX40 ligand and a second intracellular signaling domain containing the whole or a portion of any one selected from CD3-zeta and DAP12. Further, for example, the chimeric antigen receptor may include a first intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of CD3-zeta and DAP12 and a second intracellular signaling domain containing the whole or a portion of OX40 ligand. According to another embodiment of the present invention, the chimeric antigen receptor may include three intracellular signaling domains.
- the chimeric antigen receptor may include: a tirst intracellular signaling domain linked to the transmembrane domain; a second intracellular signaling domain linked to a terminal of the first intracellular signaling domain that is not linked with the transmembrane domain; and a third intracellular signaling domain linked to a terminal of the second intracellular signaling domain that is not linked with the first intracellular signaling domain.
- the first intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of 4-1BB, OX40, OX40 ligand, CD28 and DAP10
- the second intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB
- the third intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12.
- at least one of the first intracellular signaling domain, the second intracellular signaling domain and the third intracellular signaling domain may include the whole or a portion of OX40 ligand.
- the present invention may provide a chimeric antigen receptor, which includes: a first intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of CD28 and 4-1BB; a second intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB; and a third intracellular signaling domain containing the whole or a portion of CD3-zeta, wherein the first, second and third intracellular signaling domains are arranged in order from the cell membrane toward the inside of the cell.
- the above respective domains may be directly linked to one another or may be linked by a linker.
- the chimeric antigen receptor may further include: a transmembrane domain linked to the first intracellular signaling domain; a spacer domain linked to the transmembrane domain; and an extracellular domain linked to the spacer domain.
- the chimeric antigen receptor may further include a signal sequence linked to the extracellular domain.
- the above respective domains may be directly linked to one another or may be linked by a linker.
- the extracellular domain is a domain for specifically binding with an antibody or specifically recognizing an antigen, for example, an Fc receptor, an antigen-binding fragment of an antibody such as a single-chain variable fragment (ScFv), NK receptor (natural cytotoxicity receptor), NKG2D, 2B4 or DNAM-1, etc.
- an Fc receptor an antigen-binding fragment of an antibody
- ScFv single-chain variable fragment
- NK receptor natural cytotoxicity receptor
- NKG2D, 2B4 or DNAM-1 etc.
- the term “extracellular domain” is used with the same meanings as the “antigenic recognition site”, “antigen-binding fragment” and/or “antibody binding site.”
- the chimeric antigen receptor may include an Fc receptor as the extracellular domain, and therefore, can be used along with a variety of antibodies depending on cell types of cancer to be treated.
- the Fc receptor may include any one selected from the group consisting of CD16, CD32, CD64, CD23 and CD89, and variants thereof.
- the Fc receptor may include CD16 or variants thereof, and most specifically, may include the whole or a portion of CD16 V158 variant (CD16V).
- the chimeric antigen receptor of the present invention may include, as the extracellular domain, an antigen-binding fragment of an antibody which directly recognizes the antigen without co-administration along with the antibody.
- the antigen-binding fragment may be an Fab fragment, F(ab′) fragment, F(ab′)2 fragment or Fv fragment.
- the antibody may be any one of various types of antibodies capable of binding antigen-specifically.
- the antibody may be one in which one light chain and one heavy chain are bonded with each other, or one in which two light chains and two heavy chains are bonded with each other.
- the antibody when two light chains and two heavy chains are bonded with each other, the antibody may be one in which the first unit including the first light chain and the first heavy chain bonded with each other and the second unit including the second light chain and the second heavy chain bonded with each other are combined with each other.
- the bond may be a disulfide bond, but it is not limited thereto.
- the above two units may be the same as or different from each other.
- the first unit including the first light chain and the first heavy chain and the second unit including the second light chain and the second heavy chain may be the same as or different from each other.
- an antibody prepared to recognize two different antigens by the first unit and the second unit, respectively is commonly referred to as a ‘bispecific antibody’ in the related art.
- the antibody may be one in which the above three or more units are combined with one another.
- the antigen-binding fragment of the present invention may be derived from various types of antibodies as described above, but it is not limited thereto.
- the extracellular domain used herein may be a NK receptor (natural cytotoxicity receptor).
- the NK receptor may include NKp46, NKp30, NKp44, NKp80 and NKp65 receptors, but it is not limited thereto.
- the signal sequence may include the whole or a portion of CD16.
- the extracellular domain may include the whole or a portion of CD16 V158 variant (CD16V).
- the spacer domain may include the whole or a portion of any one selected from the group consisting of CD8.alpha. (CD8-alpha) and CD28.
- the transmembrane domain may include the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.
- the present invention provides immune cells (e.g., NK cells) to express the above-described chimeric antigen receptor according to the present invention.
- immune cells e.g., NK cells
- the immune cells of the present invention may exhibit toxicity to tumor cells. It is determined that the chimeric antigen receptor according to the invention exhibits specific toxicity to what types of tumor cells depending on what types of antibodies are combined with the extracellular domains. Therefore, the types of tumor cells, to which the immune cells expressing the chimeric antigen receptor according to the present invention may exhibit specific toxicity, are not particularly limited. According to one embodiment, when the immune cells (e.g., NK cells) of the present invention are used along with rituximab, the cells may exhibit toxicity to malignant lymphoma cells. For example, the malignant lymphoma cells may express CD20. Further, for example, the malignant lymphoma may be B-cell lymphoma.
- the invention further provides a pharmaceutical composition for prevention or treatment of tumor or tumor metastasis, which includes the immune cells (e.g., NK cells) expressing the above-described chimeric antigen receptor according to the present invention, in the number of 2 to 7.5 times the number of tumor cells (e.g., malignant lymphoma cells) in a subject to be treated (‘treatment target’).
- the number of immune cells (e.g., NK cells) included in the pharmaceutical composition of the present invention in a single dose may range from 0.75 to 10 times the number of tumor cells (e.g., malignant lymphoma cells) in the treatment target.
- the number of the immune cells (e.g., NK cells) in a single dose may range from 2 to 7.5 times the number of tumor cells (e.g., malignant lymphoma cells) in the treatment target.
- NK cell lines are engineered to express CAR.
- the NK cell line is NK-92.
Abstract
Disclosed are combination therapies for cancer utilizing the leverage of the adaptive immune system through chimeric antigen receptor (CAR) T cells, combined with leveraging the innate immune system by using CAR-macrophages (CAR-M) and CAR-natural killer (NK) cells. In some embodiments, the invention teaches the initial modification of the tumor microenvironment by administration of CAR-M and CAR-NK. The alteration of the tumor microenvironment results in reduction of barriers for CAR-T cells to enter the tumor, which allows for efficacy of CAR-T in treatment of solid tumors. In some embodiments adjuvant immunotherapies are utilized to expand immunological attack such as addition of complement, immunotherapeutic antibodies, chemotherapy and radiotherapy approaches.
Description
- This application claims priority to U.S. Provisional Application No. 63/400,740, titled “Combination Therapy of Solid Tumors using Chimeric Antigen Receptor Cells Representing Adaptive and Innate Immunity”, filed Aug. 24, 2022, which is hereby incorporated by reference in its entirety.
- The teachings herein relate to the use of modified immunological cells for the treatment of cancer.
- It is known that there have been a number of immunotherapeutic agents that are now used in cancer treatment, including therapeutic monoclonal antibodies (mAbs), bi-specific T-cell engagers and chimeric antigen receptors (CARs).
- The field of tumor immunology was revolutionized by the clinical entry of CAR T cells. Unfortunately, successes of this approach has been primarily limited to “liquid tumors”.
- A number of immunotherapeutic agents have been described for use in cancer treatment, including therapeutic monoclonal antibodies (mAbs), bi-specific T-cell engagers and chimeric antigen receptors (CARs). Chimeric antigen receptors are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus (binder), and a transmembrane domain connected to an endodomain which transmits T-cell activation signals. The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, which recognize a target antigen, fused via a trans-membrane domain to a signaling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. CARs have been developed against various tumor-associated antigens and many are currently undergoing clinical trials. Although CAR-T cell-mediated treatment have shown success towards compact target antigens such as CD19 or GD2, chimeric antigen receptors often to fail to signal in response to antigens with bulky extracellular domains. There is therefore a need for alternative CAR T-cell approaches, capable of killing target cells expressing a large or bulky target antigen.
- Preferred embodiments include methods of treating cancer comprising administration of innate and adaptive immune cells, wherein said immune cells have been modified with a chimeric antigen receptor.
- Preferred methods include embodiments wherein said innate immune cells are macrophages and/or an ex vivo population of CD14 expressing cells.
- Preferred methods include embodiments wherein said macrophages are M1 macrophages.
- Preferred methods include embodiments wherein is macrophages are capable of producing more nitric oxide and less arginase upon activation through TLR4 as compared to control macrophages.
- Preferred methods include embodiments wherein said macrophages express CD16.
- Preferred methods include embodiments wherein said macrophages express CD25.
- Preferred methods include embodiments wherein said macrophages express CCR7.
- Preferred methods include embodiments wherein said macrophages express CD86
- Preferred methods include embodiments wherein said macrophages express CD127.
- Preferred methods include embodiments wherein said macrophages express interleukin-1 beta receptor.
- Preferred methods include embodiments wherein said macrophages express interleukin 10 receptor.
- Preferred methods include embodiments wherein said macrophages express TNF receptor p55.
- Preferred methods include embodiments wherein said macrophages express TNF receptor p75.
- Preferred methods include embodiments wherein said macrophages express CD215.
- Preferred methods include embodiments wherein said macrophages secrete IL-1 beta.
- Preferred methods include embodiments wherein said macrophages secrete IL-6.
- Preferred methods include embodiments wherein said macrophages secrete IL-8.
- Preferred methods include embodiments wherein said macrophages secrete IL-12.
- Preferred methods include embodiments wherein said macrophages secrete IL-15.
- Preferred methods include embodiments wherein said macrophages secrete IL-17.
- Preferred methods include embodiments wherein said macrophages are engineered to express HMGB-1.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-12.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-15.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-17.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-18.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-23.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-27.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-33.
- Preferred methods include embodiments wherein said macrophages are engineered to express IL-37.
- Preferred methods include embodiments wherein said macrophages are engineered to express a chimeric antigen receptor (CAR).
- Preferred methods include embodiments wherein the CAR comprises (i) an extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain containing an intracellular signaling domain; and (b) a pharmaceutically acceptable carrier or excipient; thereby treating the cancer in the human subject.
- Preferred methods include embodiments wherein the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain, an Fc.epsilon.R intracellular signaling domain, an Fc.gamma.R intracellular signaling domain, or a TRIF intracellular signaling domain.
- Preferred methods include embodiments wherein the intracellular domain comprises two or more intracellular signaling domains.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR4 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR2 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR3 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR5 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR7/8 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR9 transmembrane domain.
- Preferred methods include embodiments wherein the extracellular domain further comprises a CD8a hinge domain.
- Preferred methods include embodiments wherein said macrophages are CD14+ cells comprises a population of CD14+/CD16+ cells.
- Preferred methods include embodiments wherein said macrophages are CD14+ cells comprises a population of CD14+/CD56+ cells.
- Preferred methods include embodiments wherein said macrophages comprise a population of CD14+ monocytes, a population of CD14+ macrophages or a population of CD14+ dendritic cells.
- Preferred methods include embodiments wherein said macrophages are CD14+ cells that are derived from the human subject.
- Preferred methods include embodiments wherein the population of CD14+ cells is obtained from a leukapheresis sample, a blood sample, or a PBMC sample from the human subject.
- Preferred methods include embodiments wherein the population of CD14+ cells is an ex vivo population of virally transduced cells.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells comprises a viral component.
- Preferred methods include embodiments wherein the antigen binding domain is a single domain antibody (sdAb) or a single chain variable fragment (scFv).
- Preferred methods include embodiments wherein the antigen binding domain is an anti-HER2/neu binding domain.
- Preferred methods include embodiments wherein the sequence of the recombinant polynucleic acid encoding the CAR is from a viral vector.
- Preferred methods include embodiments wherein the method further comprises transducing a viral vector into a population of CD14+ cells ex vivo, thereby obtaining the ex vivo population of CD14+ cells comprising the recombinant polynucleic acid with a sequence encoding a CAR.
- Preferred methods include embodiments wherein the method comprises (i) extracting a blood sample from the human subject; (ii) isolating monocytes from the blood sample; and (iii) transfecting the monocytes from the blood sample with the recombinant polynucleic acid with a sequence encoding a CAR; and wherein administering comprises infusing.
- Preferred methods include embodiments wherein the recombinant polynucleic acid is mRNA.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells stimulates killing of cancer cells in the human subject by T cells of the human subject.
- Preferred methods include embodiments wherein the intracellular domain of the CAR is capable of inducing monocytic differentiation to M1 macrophages in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells enhances or improves effector function of a T cell in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells directly kills cancer cells in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells inhibits macrophage or macrophage related cells of the human subject from promoting tumor growth.
- Preferred methods include embodiments wherein the ex vivo population of CD14+ cells is phagocytic.
- Preferred methods include embodiments wherein the cancer is a lymphoma.
- Preferred methods include embodiments wherein the cancer is a solid tumor.
- Preferred methods include embodiments wherein the cancer is a breast cancer.
- Preferred methods include embodiments wherein the cancer is a metastatic cancer.
- Preferred methods include embodiments wherein the cancer is an ErbB-2-expressing cancer.
- Preferred methods include embodiments wherein the method further comprises administering GM-CSF, IL-2, an agent that blocks CD47 activity or an agent that induces immunogenic cell death to the human subject.
- Preferred methods include embodiments wherein the method further comprises administering G-CSF.
- Preferred methods include embodiments wherein the method further comprises administering M-CSF.
- Preferred methods include embodiments wherein the method further comprises administering IL-2.
- Preferred methods include embodiments wherein the method further comprises administering antibody to interleukin-10.
- Preferred methods include embodiments wherein the method further comprises administering TNF-alpha.
- Preferred methods include embodiments wherein the method further comprises administering lymphotoxin.
- Preferred methods include embodiments wherein the method further comprises administering interferon alpha.
- Preferred methods include embodiments wherein the method further comprises administering interferon beta.
- Preferred methods include embodiments wherein the method further comprises administering interferon gamma.
- Preferred methods include embodiments wherein the method further comprises administering interferon tau.
- Preferred methods include embodiments wherein the method further comprises administering interferon omega.
- Preferred methods include embodiments wherein the method further comprises administering IL-7.
- Preferred methods include embodiments wherein the method further comprises administering IL-9.
- Preferred methods include embodiments wherein the method further comprises administering IL-12.
- Preferred methods include embodiments wherein the method further comprises administering IL-15.
- Preferred methods include embodiments wherein the method further comprises administering IL-18.
- Preferred methods include embodiments wherein the method further comprises administering IL-23.
- Preferred methods include embodiments wherein the method further comprises administering IL-33.
- Preferred methods include embodiments wherein the method further comprises administering C5 component of complement.
- Preferred methods include embodiments wherein the method further comprises administering C3 component of complement.
- Preferred methods include embodiments wherein the method further comprises administering BCG.
- Preferred methods include embodiments wherein the method further comprises administering a chemotherapeutic agent.
- Preferred methods include embodiments wherein the method further comprises administering an immunotherapeutic agent.
- Preferred methods include embodiments wherein the method further comprises administering an immunotherapeutic agent.
- Preferred methods include embodiments wherein the method further comprises administering radiotherapy.
- Preferred methods include embodiments wherein said innate immune cell is a natural killer (NK) cell.
- Preferred methods include embodiments wherein said NK cell is cytotoxic towards K562 cells.
- Preferred methods include embodiments wherein said NK cell expresses CD25.
- Preferred methods include embodiments wherein said NK cell expresses CD69.
- Preferred methods include embodiments wherein said NK cell expresses CD133.
- Preferred methods include embodiments wherein said NK cell expresses CD56.
- Preferred methods include embodiments wherein said NK cell expresses CD16.
- Preferred methods include embodiments wherein said NK cell expresses CD57.
- Preferred methods include embodiments wherein said NK cell expresses Fas ligand.
- Preferred methods include embodiments wherein said NK cell expresses IL-7 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-3 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-10 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-12 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-15 receptor.
- Preferred methods include embodiments wherein said NK cell expresses IL-18 receptor.
- Preferred methods include embodiments wherein said NK cells are derived from peripheral blood.
- Preferred methods include embodiments wherein said NK cells are derived from umbilical cord blood.
- Preferred methods include embodiments wherein said NK cells are derived from menstrual blood.
- Preferred methods include embodiments wherein said NK cells are derived from bone marrow.
- Preferred methods include embodiments wherein said NK cells are derived from mobilized peripheral blood.
- Preferred methods include embodiments wherein mobilization of blood is achieved by treatment of the patient with agents that increase oxidative stress.
- Preferred methods include embodiments wherein said agents that increase oxidative stress are ozone.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with G-CSF.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with GM-CSF.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with M-CSF.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with mozibil.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with VEGF.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with thrombopoietin.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with TNF-alpha.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with hepatocyte growth factor.
- Preferred methods include embodiments wherein said mobilization is accomplished by treating the patient with flt-3 ligand.
- Preferred methods include embodiments wherein said NK cells are generated from progenitor cells.
- Preferred methods include embodiments wherein said progenitors are umbilical cord derived.
- Preferred methods include embodiments wherein said progenitors are placental derived.
- Preferred methods include embodiments wherein said progenitors are CD34 derived.
- Preferred methods include embodiments wherein said progenitors are CD133 derived.
- Preferred methods include embodiments wherein said progenitors are inducible pluripotent stem cell derived.
- Preferred methods include embodiments wherein said progenitors are embryonic stem cell derived.
- Preferred methods include embodiments wherein said progenitors are parthenogenic stem cell derived.
- Preferred methods include embodiments wherein said progenitors are lymphoid progenitor cell derived.
- Preferred methods include embodiments wherein said NK cells are engineered to express a chimeric antigen receptor (CAR).
- Preferred methods include embodiments wherein the CAR comprises (i) an extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain containing an intracellular signaling domain; and (b) a pharmaceutically acceptable carrier or excipient; thereby treating the cancer in the human subject.
- Preferred methods include embodiments wherein the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain, an Fc.epsilon.R intracellular signaling domain, an Fc.gamma.R intracellular signaling domain, or a TRIF intracellular signaling domain.
- Preferred methods include embodiments wherein the intracellular domain comprises two or more intracellular signaling domains.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR4 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR2 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR3 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR5 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR7/8 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR9 transmembrane domain.
- Preferred methods include embodiments wherein the extracellular domain further comprises a CD8a hinge domain.
- Preferred methods include embodiments wherein said NK are pre-activated by culture in IL-2.
- Preferred methods include embodiments wherein said NK cells are pre-activated by culture with dendritic cells.
- Preferred methods include embodiments wherein said NK cells are pre-activated by culture with monocytes.
- Preferred methods include embodiments wherein said NK cells are capable of proliferating approximately one multiplication ever 15-36 hours.
- Preferred methods include embodiments wherein said NK cells are capable of proliferating approximately one multiplication ever 20-30 hours.
- Preferred methods include embodiments wherein said NK cells are capable of proliferating approximately one multiplication ever 15-36 hours.
- Preferred methods include embodiments wherein NK cells are virally transfected.
- Preferred methods include embodiments wherein the antigen binding domain is a single domain antibody (sdAb) or a single chain variable fragment (scFv).
- Preferred methods include embodiments wherein the antigen binding domain is an anti-HER2/neu binding domain.
- Preferred methods include embodiments wherein the sequence of the recombinant polynucleic acid encoding the CAR is from a viral vector.
- Preferred methods include embodiments wherein the method further comprises transducing a viral vector into a population of CD56+ cells ex vivo, thereby obtaining the ex vivo population of CD56+ cells comprising the recombinant polynucleic acid with a sequence encoding a CAR.
- Preferred methods include embodiments wherein the method comprises (i) extracting a blood sample from the human subject; (ii) isolating NK cells from the blood sample; and (iii) transfecting the NK cells from the blood sample with the recombinant polynucleic acid with a sequence encoding a CAR; and wherein administering comprises infusing.
- Preferred methods include embodiments wherein the recombinant polynucleic acid is mRNA.
- Preferred methods include embodiments wherein the ex vivo population of CD56+ cells stimulates killing of cancer cells in the human subject by T cells of the human subject.
- Preferred methods include embodiments wherein the intracellular domain of the CAR is capable of inducing activation of NK cell in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD56+ cell enhances or improves effector function of a T cell in the human subject.
- Preferred methods include embodiments wherein the ex vivo population of CD56+ cells directly kills cancer cells in the human subject.
- Preferred methods include embodiments wherein adaptive immune cell is a T cell.
- Preferred methods include embodiments wherein said T cell is a CD4 T cell.
- Preferred methods include embodiments wherein said T cell is a CD8 T cell.
- Preferred methods include embodiments wherein said T cell is an NKT cell.
- Preferred methods include embodiments wherein said T cell is a double negative T cell.
- Preferred methods include embodiments wherein said T cell expresses interleukin-2 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-4 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-7 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-9 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-12 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-15 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-17 receptor.
- Preferred methods include embodiments wherein said T cell expresses interleukin-18 receptor.
- Preferred methods include embodiments wherein said NK cells are engineered to express a chimeric antigen receptor (CAR).
- Preferred methods include embodiments wherein the CAR comprises (i) an extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain containing an intracellular signaling domain; and (b) a pharmaceutically acceptable carrier or excipient; thereby treating the cancer in the human subject.
- Preferred methods include embodiments wherein the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain, an Fc.epsilon.R intracellular signaling domain, an Fc.gamma.R intracellular signaling domain, or a TRIF intracellular signaling domain.
- Preferred methods include embodiments wherein the intracellular domain comprises two or more intracellular signaling domains.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR4 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR2 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR3 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR5 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR7/8 transmembrane domain.
- Preferred methods include embodiments wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR9 transmembrane domain.
- Preferred methods include embodiments wherein the extracellular domain further comprises a CD8a hinge domain.
- Preferred methods include embodiments wherein said T cells are pre-activated by culture in IL-2.
- Preferred methods include embodiments wherein said T cells are pre-activated by culture with dendritic cells.
- Preferred methods include embodiments wherein said T cells are pre-activated by culture with monocytes.
- Preferred methods include embodiments wherein said T cells are capable of proliferating approximately one multiplication ever 15-36 hours.
- Preferred methods include embodiments wherein said T cells are capable of proliferating approximately one multiplication ever 20-30 hours.
- Preferred methods include embodiments wherein said T cells are capable of proliferating approximately one multiplication ever 15-36 hours.
- Preferred methods include embodiments wherein T cells are virally transfected.
- Preferred methods include embodiments wherein the antigen binding domain is a single domain antibody (sdAb) or a single chain variable fragment (scFv).
- Preferred methods include embodiments wherein the antigen binding domain is an anti-HER2/neu binding domain.
- Preferred methods include embodiments wherein the sequence of the recombinant polynucleic acid encoding the CAR is from a viral vector.
- Preferred methods include embodiments wherein CAR-macrophages are injected initially, alone or in combination with CAR-NK.
- Preferred methods include embodiments wherein CAR-T cells are administered at a timepoint after which CAR-macrophages and/or CAR-NK have modified to tumor microenvironment to allow for CAR-T cells to enter said tumor microenvironment.
- Preferred methods include embodiments wherein all cells expressing CAR possess an inhibitor of NR2F6.
- The invention provides means of treating cancer through inactivating the tumor microenvironment followed by administration of CAR-T cells. The invention provides the use of CAR-Macrophages and CAR-NK cells for manipulation of the tumor microenvironment in order to decrease immunesuppressinvess and thus allowing for entry of CAR-T cells into solid tumors. The role of the CAR-macrophages and CAR-NK cells are to increase propensity for Th1 immunity and allow for activity of CAR-T cells which otherwise is blunted by mediators such as PGE-2, IL-10, TGF-beta and soluble TNF-alpha receptor found in the tumor microenvironment.
- The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
- As used herein, terms “comprise,” “have,” “has,” and “include” and their conjugates, as used herein, mean “including but not limited to.” While various compositions, and methods are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions, methods, and devices can also “consist essentially of” or “consist of” the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.
- “Activation”, as used herein, refers to the state of a MIL that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be as sociated with induced cytokine production, and detectable effector functions. The term “activated MILs” refers to, among other things, MILs that are undergoing cell division.
- The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.
- The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.
- The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the embodiments include, but are not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
- The term “anti-tumor effect” as used herein, refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies to prevent the occurrence of tumor in the first place.
- The term “auto-antigen” means any self-antigen which is mistakenly recognized by the immune system as being foreign. Auto-antigens comprise, but are not limited to, cellular proteins, phosphoproteins, cellular surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.
- As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
- “Allogeneic” refers to a graft derived from a different animal of the same species.
- “Xenogeneic” refers to a graft derived from an animal of a different species.
- The term “cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may include non-solid tumors (such as hematological tumors, for example, myeloma, leukemias and lymphomas) or may include solid tumors. Types of cancers to be treated with the CARs as described herein include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.
- “Co-stimulatory ligand,” as the term is used herein, includes a molecule on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a MIL, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a MIL response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a MIL, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
- A “co-stimulatory molecule” refers to the cognate binding partner on a MIL that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the MIL, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.
- A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to MIL proliferation and/or upregulation or downregulation of key molecules.
- A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
- An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.
- “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
- As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.
- As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.
- The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
- “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
- “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared .times.100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
- The term “immunoglobulin” or “Ig,” as used herein is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE.
- “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
- As used herein, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
- A “Lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
- Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
- The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
- The term “overexpressed” tumor antigen or “overexpression” of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.
- “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
- The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
- As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
- The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
- A classical chimeric antigen receptor (CAR) is a chimeric type I trans-membrane protein which connects an extracellular antigen-recognizing domain (binder) to an intracellular signaling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of IgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the IgG1 hinge alone, depending on the antigen. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain. Early CAR designs had endodomains derived from the intracellular parts of either the .gamma. chain of the Fc.epsilon.R1 or CD3.zeta. Consequently, these first generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3.zeta. results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal—namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related OX40 and 41 BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards tumor cells expressing the targeted antigen. CARs typically therefore comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain which comprises or associates with a signaling domain. A CAR may have the general structure: Antigen binding domain-spacer domain-transmembrane domain-intracellular signaling domain (endodomain). Within the context of the current invention, CARs may be utilized for application in innate cells, such as NK and macrophages, as well as in their more classical embodiment, T cells.
- The invention provides means of inducing an anti-cancer response in a mammal, comprising the steps of initially “priming” the mammal by administering an agent that causes local accumulation of CAR-macrophage. Subsequently, a tumor antigen is administered in the local area where said agents causing accumulation of antigen presenting cells is administered. A time period is allowed to pass to allow for said antigen presenting cells to traffic to the lymph nodes. Subsequently a maturation signal, or a plurality of maturation signals are administered to enhance the ability of said antigen presenting cell to activate adaptive immunity. In some embodiments of the invention activators of adaptive immunity are concurrently given, as well as inhibitors of the tumor derived inhibitors are administered to derepress the immune system.
- In one embodiment priming of the patient is achieved by administration of GM-CSF subcutaneously in the area in which antigen is to be injected. Various scenarios are known in the art for administration of GM-CSF prior to administration, or concurrently with administration of antigen. The practitioner of the invention is referred to the following publications for dosage regimens of GM-CSF and also of peptide antigens. Subsequent to priming, the invention calls for administration of tumor antigen. Various tumor antigens may be utilized, in one preferred embodiment, lysed tumor cells from the same patient area utilized. Means for generation of lysed tumor cells are well known in the art and described in the following references. One example method for generation of tumor lysate involves obtaining frozen autologous samples which are placed in hanks buffered saline solution (HBSS) and gentamycin 50.mu.g/ml followed by homogenization by a glass homogenizer. After repeated freezing and thawing, particle-containing samples are selected and frozen in aliquots after radiation with 25 kGy. Quality assessment for sterility and endotoxin content is performed before freezing. Cell lysates are subsequently administered into the patient in a preferred manner subcutaneously at the local areas where DC priming was initiated. After 12-72 hours, the patient is subsequently administered with an agent capable of inducing maturation of DC. Agents useful for the practice of the invention, in a preferred embodiment include BCG and HMGB1 peptide. Other useful agents include: a) histone DNA; b) imiqimod; c) beta-glucan; d) hsp65; e) hsp90; 0 HMGB-1; g) lipopolysaccharide; h) Pam3CSK4; i) Poly I: Poly C; j) Flagellin; k) MALP-2; 1) Imidazoquinoline; m) Resiquimod; n) CpG oligonucleotides; o) zymosan; p) peptidoglycan; q) lipoteichoic acid; r) lipoprotein from gram-positive bacteria; s) lipoarabinomannan from mycobacteria; t) Polyadenylic-polyuridylic acid; u) monophosphoryl lipid A; v) single stranded RNA; w) double stranded RNA; x) 852A; y) rintatolimod; z) Gardiquimod; and aa) lipopolysaccharide peptides. The procedure is performed in a preferred embodiment with the administration of IDO silencing siRNA or shRNA containing the effector sequences a) UUAUAAUGACUGGAUGUUC; b) GUCUGGUGUAUGAAGGGUU; c) CUCCUAUUUUGGUUUAUGC and d) GCAGCGUCUUUCAGUGCUU. siRNA or shRNA may be administered through various modalities including biodegradable matrices, pressure gradients or viral transfect. In another embodiment, autologous dendritic cells are generated and IDO is silenced, prior to, concurrent with or subsequent to silencing, said dendritic cells are pulsed with tumor antigen and administered systemically.
- In one embodiment of the invention mature DC are modified with CAR transfection prior to administration. Culture of dendritic cells is well known in the art, for example, U.S. Pat. No. 6,936,468, issued to Robbins, et al., for the use of tolerogenic dendritic cells for enhancing tolerogenicity in a host and methods for making the same. Although the current invention aims to reduce tolerogenesis, the essential means of dendritic cell generation are disclosed in the patent. U.S. Pat. No. 6,734,014, issued to Hwu, et al., for methods and compositions for transforming dendritic cells and activating T cells. Briefly, recombinant dendritic cells are made by transforming a stem cell and differentiating the stem cell into a dendritic cell. The resulting dendritic cell is said to be an antigen presenting cell which activates T cells against MHC class I-antigen targets. Antigens for use in dendritic cell loading are taught in, e.g., U.S. Pat. No. 6,602,709, issued to Albert, et al. This patent teaches methods for use of apoptotic cells to deliver antigen to dendritic cells for induction or tolerization of T cells. The methods and compositions are said to be useful for delivering antigens to dendritic cells that are useful for inducing antigen-specific cytotoxic T lymphocytes and T helper cells. The disclosure includes assays for evaluating the activity of cytotoxic T lymphocytes. The antigens targeted to dendritic cells are apoptotic cells that may also be modified to express non-native antigens for presentation to the dendritic cells. The dendritic cells are said to be primed by the apoptotic cells (and fragments thereof) capable of processing and presenting the processed antigen and inducing cytotoxic T lymphocyte activity or may also be used in vaccine therapies. U.S. Pat. No. 6,455,299, issued to Steinman, et al., teaches methods of use for viral vectors to deliver antigen to dendritic cells. Methods and compositions are said to be useful for delivering antigens to dendritic cells, which are then useful for inducing T antigen specific cytotoxic T lymphocytes. The disclosure provides assays for evaluating the activity of cytotoxic T lymphocytes. Antigens are provided to dendritic cells using a viral vector such as influenza virus that may be modified to express non-native antigens for presentation to the dendritic cells. The dendritic cells are infected with the vector and are said to be capable of presenting the antigen and inducing cytotoxic T lymphocyte activity or may also be used as vaccines. Immune cells for use in the practice of the invention include DCs, the presence of which may be checked in the previously described method, are preferably selected from myeloid cells (such as monocytic cells and macrophages) expressing langerin, MHC (major histocompatibility complex) class II, CCR2 (chemokine (C-C motif) receptor 2), CX3CR1 and/or Grl molecules in mice; myeloid cells expressing CD14, CD16, HLA dR (human leukocyte antigen disease resistance) molecule, langerin, CCR2 and/or CX3CR1 in humans; dendritic cells expressing CD11c, MHC class II molecules, and/or CCR7 molecules; and IL-1.beta. producing dendritic cells. CD8 T cells, the presence of which may be checked in the previously described method, are preferably selected from CD3+, CD4+ and/or CD8+T lymphocytes, FOXP3 (forkhead box P3) T lymphocytes, Granzyme B/TIA (Tcell-restricted intracellular antigen) T lymphocytes, and Tc1 cells (IFN-.gamma. producing CD8+T lymphocytes). Immune cells expressing a protein that binds calreticulin, such immune cells may be selected from cells expressing at least one of the following proteins: LRP1 (Low density lipoprotein receptor-related protein 1, CD91), Ca.sup.++-binding proteins such as SCARF1 and SCARF2, MSR1 (Macrophage scavenger receptor 1), SRA, CD59 (protectin), CD207 (langerin), and THSDI (thrombospondin). There are numerous means known in the art to identify cells expressing various antigens, these include immunochemistry, immunophenotyping, flow cytometry, Elispot assays, classical tetramer staining, and intracellular cytokine staining.
- Macrophages selectively phagocytose tumor cells, but this process is countered by protective molecules on tumor cells such as CD47, which binds macrophage signal-regulatory protein a to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. In one embodiment of the invention CAR-MSC are administered together with an agent that blocks CD47 activity. It has been demonstrated that activation of TLR signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance tumor phagocytosis. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be a protein found on cancer cells that activated macrophage phagocytosis of tumors, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin. In one embodiment of the invention TLR agonists are administered that stimulate expression of calreticulin and/or enhance macrophage phagocytosis of tumors. IL-27 induces macrophage ability to kill tumor cells in vitro and in vivo, as well as altering the tumor promoting M2/myeloid suppressor cells into tumoricidal cells. In one embodiment of the invention addition of IL-27 or compounds capable of activating the IL-27 receptor signaling are administered together with IL-27 to enhance tumor phagocytosis by macrophages.
- Tumor-associated macrophages, deriving from monocytes or migrating into the tumor, are an important constituent of tumor microenvironments, which in many cases modulates tumor growth, tumor angiogenesis, immune suppression, metastasis and chemoresistance. Mechanisms of macrophage promotion of tumor growth include production of EGF, M-CSF, and VEGF. Macrophage infiltration of tumors is associated with poor prognosis in renal, melanoma, breast, pancreatic, lung, endometrial, bladder, prostate. Tumor growth are inhibited when monocytes/macrophages are ablated. There is ample evidence that many anticancer modalities currently used in the clinic have unique and distinct properties that modulate the recruitment, polarization and tumorigenic activities of macrophages in the tumor microenvironments. By manipulating tumor-associated macrophages significant impact on the clinical efficacies of and resistance to these anticancer modalities. Accordingly, in one aspect of the invention, CAR-macrophages are utilized to force the tumor microenvironment to stimulate tumor killing and inhibit macrophage or macrophage related cells from promoting tumor growth. Within the context of the invention, the use of drugs targeting tumor-associated macrophages, especially c-Fms kinase inhibitors and humanized antibodies targeting colony-stimulating factor-1 receptor, are envisioned.
- Tumors mediate various effects to reprogram macrophages, these are usually mediated via IL-10 and other cytokines such as VEGF, TGF-beta, and M-CSF, which cause macrophages to lose tumor cytotoxicity and shift into tumor promoting, immune suppressive, angiogenic supporting cells. Related to tumor manipulated monocytes are myeloid derived suppressor cells, which are similar to myeloid progenitor cells, or the previously described “natural suppressor” cell.
- Irradiated tissues induce a TLR-1 reprogramming of macrophages to promote tumor growth and angiogenesis. Macrophage promotion of tumor growth is seen in numerous situations, in one example, treating of tumor bearing animals with BRAF inhibitors results in upregulation of macrophage production of VEGF which accelerates tumor growth. Mechanistically, it is known that tumors produce factors such as GM-CSF which in part stimulate macrophages to produce CCL18, which promotes tumor metastasis. Additionally, the lactic acid microenvironment of the tumor has been shown to promote skewing of macrophages towards at tumor-promoting M2 type. It has been shown that lactic acid produced by tumor cells, as a by-product of aerobic or anaerobic glycolysis, possesses an essential role in inducing the expression of VEGF and the M2-like polarization of tumor-associated macrophages, specifically inducing expression of arginase 1 through a HIF-1alpha dependent pathway. Mechanistically, it is known that lactic acid in tumors is generated in a large part by lactate dehydrogenase-A (LDH-A), which converts pyruvate to lactate. siRNA silencing of LDH-A in Pan02 pancreatic cancer cells that are injected in C57BL/6 mice results in development of smaller tumors than mice injected with wild type, non-silenced Pan02 cells. Associated with the reduced tumor growth were observations of a decrease in the frequency of myeloid-derived suppressor cells (MDSCs) in the spleens of mice carrying LDH-A-silenced tumors. NK cells from LDH-A-depleted tumors had improved cytolytic function. Exogenous lactate administration was shown to increase the frequency of MDSCs generated from mouse bone marrow cells with GM-CSF and IL-6 in vitro. Furthermore, lactate pretreatment of NK cells in vitro inhibited cytolytic function of both human and mouse NK cells. This reduction of NK cytotoxic activity was accompanied by lower expression of perforin and granzyme in NK cells. The expression of NKp46 was lower in lactate-treated NK cells. Accordingly, in one embodiment of the invention, depletion of glucose levels using a ketogenic diet to lower lactate production by glycolytic tumors is utilized to augment therapeutic effects of CAR-macrophage. Utilization of ketogenic diet has been previously described for immune modulation, and cancer therapy. Specific quantification of intratumoral lactate and its manipulation has been described and incorporated by reference. Potentiation of chemotherapeutic and radiotherapeutic effects by ketogenic diets have been reported and techniques are incorporated by reference for use with the current CAR-macrophage invention. Suppression of tumor growth and activity induced by ketogenic diet may be augmented by addition of hyperbaric oxygen, thus in one embodiment of the invention, the utilization of oxidative therapies, as disclosed in references incorporated, together with ketogenic diet is utilized to augment therapeutic efficacy of CAR-macrophage.
- Not only has it been well known that monocytes and macrophages infiltrate tumors and appear to support tumor growth through growth factor production and secretion of angiogenic agents, but suggestions have been made that tumors themselves, as part of the epithelial mesenchymal transition may actually differentiate into monocytes in part associated with TGF-beta production. Specifically, a study reported performing gene-profiling analysis of mouse mammary EpRas tumor cells that had been allowed to adopt an epithelial to mesenchymal transition program after long-term treatment with TGF-.beta.1 for 2 weeks. While the treated cells acquired traits of mesenchymal cell differentiation and migration, gene analysis revealed another cluster of induced genes, which was specifically enriched in monocyte-derived macrophages, mast cells, and myeloid dendritic cells, but less in other types of immune cells. Further studies revealed that this monocyte/macrophage gene cluster was enriched in human breast cancer cell lines displaying an EMT or a Basal B profile, and in human breast tumors with EMT and undifferentiated (ER-/PR-) characteristics. The plasticity of tumor cells to potentially monocytic lineages should come as no surprise given that tumor cells have been shown to differentiate directly into pericytes, and endothelial cells/vascular channels. Dopamine possesses antiangiogenic effects as well as myeloprotective effects, in one embodiment of the invention addition of dopamine to the CAR-macrophage treatment is disclosed. Vinblastine is a widely used chemotherapeutic agent that has been demonstrated to induce dendritic cell maturation. In one embodiment of the invention CAR-macrophage are utilized together with vinblastine therapy to induce augmented anticancer activity. Oxiplatin and anthracyclines have been demonstrated to not only directly kill tumor cells but also stimulate T cell immunity against tumor cells. It was demonstrated that these agents induce a rapid and prominent invasion of interleukin (IL)-17-producing .gamma..delta. (V.gamma.4(+) and V.gamma.6(+)) T lymphocytes (.gamma.6 T17 cells) that precedes the accumulation of CD8 CTLs within the tumor bed. In T cell receptor .delta.(−/−) or V.gamma.4/6(−/−) mice, the therapeutic efficacy of chemotherapy was reduced and furthermore no IL-17 was produced by tumor-infiltrating T cells, and CD8 CTLs did not invade the tumor after treatment. Although .gamma..delta. Th17 cells could produce both IL-17A and IL-22, the absence of a functional IL-17A-IL-17R pathway significantly reduced tumor-specific T cell responses elicited by tumor cell death, and the efficacy of chemotherapy in four independent transplantable tumor models. The adoptive transfer of .gamma..delta. T cells to naive mice restored the efficacy of chemotherapy in IL-17A(−/−) hosts. The anticancer effect of infused .gamma..delta. T cells was lost when they lacked either IL-1R1 or IL-17A. Intratumoral injection of dendritic cells stimulates antitumor immunity in vivo in clinical situations, suggesting that modulating the antigen presenting cell in the tumor microenvironment will induce an antitumor response. Administration of radiotherapy to tumors to induce immunogenic cell death, followed by intratumoral administration of DC has been demonstrated to result in enhanced antigen presentation, accordingly, this technique may be modified to enhance effects of CAR-macrophages. The induction of immunity to tumors in the present invention is associated with the unique nature of: a) ongoing basal cell death within the tumor; and b) cell death induced by chemotherapy, radiotherapy, hyperthermia, or otherwise induced cell death. Cell death can be classified according to the morphological appearance of the lethal process (that may be apoptotic, necrotic, autophagic or associated with mitosis), enzymological criteria (with and without the involvement of nucleases or distinct classes of proteases, like caspases), functional aspects (programmed or accidental, physiological or pathological) or immunological characteristics (immunogenic or non-immunogenic). Cell death is defined as “immunogenic” or “immune stimulatory” if dying cells that express a specific antigen (for example a tumor associated antigen, phosphotidyl serine, or calreticulin), yet are uninfected (and hence lack pathogen-associated molecular patterns), and are injected subcutaneously into mice, in the absence of any adjuvant, cause a protective immune response against said specific antigen. Such a protective immune response precludes the growth of living transformed cells expressing the specific antigen injected into mice. When cancer cells succumb to an immunogenic cell death (or immunogenic apoptosis) modality, they stimulate the immune system, which then mounts a therapeutic anti-cancer immune response and contributes to the eradication of residual tumor cells. Conversely, when cancer cells succumb to a non-immunogenic death modality, they fail to elicit such a protective immune response. Regardless of the types of cell death that are ongoing, the tumor derived immune suppressive molecules contribute to general inhibition or inability of the tumor to be eliminated. Within the practice of the invention, CAR-macrophage are administered concurrently, prior to, or subsequent to administration of an agent that induces immunogenic cell death in a patient. Methods of determining whether compounds induce immunogenic cell death are known in the art and include the following, which was described by Zitvogel et al. (a) treating the cells, the mammalian cells and inducing the cell death or apoptosis, typically of mammalian cancer cells capable of expressing calreticulin (CRT), by exposing said mammalian cells to a particular drug (the test drug), for example 18 hours; (b) inoculating (for example intradermally) the dying mammalian cells from step (a) in a particular area (for example a flank) of the mammal, typically a mouse, to induce an immune response in this area of the mammal; (c) inoculating (for example intradermally) the minimal tumorigenic dose of syngeneic live tumor cells in a distinct area (for example the opposite flank) from the same mammal, for example 7 days after step (b); and (d) comparing the size of the tumor in the inoculated mammal with a control mammal also exposed to the minimal tumorigenic dose of syngeneic live tumor cells of step (c) [for example a mouse devoid of T lymphocyte], the stabilization or regression of the tumor in the inoculated mammal being indicative of the drug immunogenicity. Other in vitro means are available for assessing the ability of various drugs or therapeutic approaches to induce immunogenic cell death. Specific characteristics to assess when screening for immunogenic cell death include: a) ability to induce dendritic cell maturation in vitro; b) ability to activate NK cells; and c) ability to induce activation of gamma delta T cells or NKT cells. Specific drugs known to induce immunogenic cell death include oxiplatine and anthracyclines, as well as radiotherapy, and hyperthermia. In the case of chemotherapies, certain chemotherapies that activate TLR4 through induction of HMGB1 have been observed to function suboptimally in patients that have a TLR4 polymorphism, thus suggesting actual contribution of TLR activation as a means of chemotherapy inhibition of cancer. Additionally, oncoviruses or oncolytic viruses are known to induce immunogenic cell death and may be useful for the practice of the invention. The CAR-macrophage disclosed in the invention may be utilized in combination with conventional immune modulators including BCG, CpG DNA, interferon alpha, tumor bacterial therapy, checkpoint inhibitors, Treg depleting agents, and low dose cyclophosphamide.
- In one embodiment of the invention CAR-macrophage cells are generated with specificity towards ROBO-4. Numerous means of generating CAR-T cells are known in the art, which are applied to CAR-macrophage. In one embodiment of the invention FMC63-28z CAR (Genebank identifier HM852952.1), is used as the template for the CAR except the anti-CD19, single-chain variable fragment sequence is replaced with an ROBO-4 fragment. The construct is synthesized and inserted into a pLNCX retroviral vector. Retroviruses encoding the ROBO-4-specific CAR are generated using the retrovirus packaging kit, Ampho (Takara), following the manufacturer's protocol. For generation of CAR-MACROPHAGE cells donor blood is obtained and after centrifugation on Ficoll-Hypaque density gradients (Sigma-Aldrich), PBMCs are plated at 2.times.10(6) cells/mL in cell culture for 2 hours and the adherent cells are collected. The cells were then stimulated for 2 days on a tissue-culture-treated 24-well plate containing M-CSF at a concentration of 100 ng/ml For retrovirus transduction, a 24-well plate are coated with RetroNectin (Takara) at 4.degree. C. overnight, according to the manufacturer's protocol, and then blocked with 2% BSA at room temperature for 30 min. The plate was then loaded with retrovirus supernatants at 300.mu.L/well and incubated at 37.degree. C. for 6 h. Next, 1.times.10(6) stimulated adherent cells in 1 mL of medium are added to 1 mL of retrovirus supernatants before being transferred to the pre-coated wells and cultured at 37.degree. C. for 2 d. The cells are then transferred to a tissue-culture-treated plate at 1.times.10 (6) cells/mL and cultured in the presence of 100 U/mL of recombinant human M-CSF, applying the T cell protocol but not utilizing IL-2 or antiCD3/antiCD28.
- Other means of generating CARs are known in the art and incorporated by reference. For example, Groner's group genetically modified T lymphocytes and endowed them with the ability to specifically recognize cancer cells. Tumor cells overexpressing the ErbB-2 receptor served as a model. The target cell recognition specificity was conferred to T lymphocytes by transduction of a chimeric gene encoding the zeta-chain of the TCR and a single chain antibody (scFv(FRP5)) directed against the human ErbB-2 receptor. The chimeric scFv(FRP5)-zeta gene was introduced into primary mouse T lymphocytes via retroviral gene transfer. Naive T lymphocytes were activated and infected by cocultivation with a retrovirus-producing packaging cell line. The scFv(FRP5)-zeta fusion gene was expressed in >75% of the T cells. These T cells lysed ErbB-2-expressing target cells in vitro with high specificity. In a syngeneic mouse model, mice were treated with autologous, transduced T cells. The adoptively transferred scFv(FRP5)-zeta-expressing T cells caused total regression of ErbB-2-expressing tumors. The presence of the transduced T lymphocytes in the tumor tissue was monitored. No humoral response directed against the transduced T cells was observed. Abs directed against the ErbB-2 receptor were detected upon tumor lysis. Hornbach et al. constructed an anti-CEA chimeric receptor whose extracellular moiety is composed of a humanized scFv derived from the anti-CEA mAb BW431/26 and the CH2/CH3 constant domains of human IgG. The intracellular moiety consists of the gamma-signaling chain of the human Fc epsilon RI receptor constituting a completely humanized chimeric receptor. After transfection, the humBW431/26 scFv-CH2CH3-gamma receptor is expressed as a homodimer on the surface of MD45 T cells. Co-incubation with CEA+ tumor cells specifically activates grafted MD45 T cells indicated by IL-2 secretion and cytolytic activity against CEA+ tumor cells. Notably, the efficacy of receptor-mediated activation is not affected by soluble CEA up to 25 micrograms/ml demonstrating the usefulness of this chimeric receptor for specific cellular activation by membrane-bound CEA even in the presence of high concentrations of CEA, as found in patients during progression of the disease (200). These methods are described to guide one of skill in the art to practicing the invention, which in one embodiment is the utilization of CAR T cell approaches towards targeting tumor endothelium as comparted to simply targeting the tumor itself.
- Targeting of mucins associated with cancers has been performed with CAR T cells by grafting the antibody that binds to the mucin with CD3 zeta chain. For the purpose of the invention, this procedure is modified for CAR-macrophage. In an older publication chimeric immune receptor consisting of an extracellular antigen-binding domain derived from the CC49 humanized single-chain antibody, linked to the CD3zeta signaling domain of the T cell receptor, was generated (CC49-zeta). This receptor binds to TAG-72, a mucin antigen expressed by most human adenocarcinomas. CC49-zeta was expressed in CD4+ and CD8+ T cells and induced cytokine production on stimulation. Human T cells expressing CC49-zeta recognized and killed tumor cell lines and primary tumor cells expressing TAG-72. CC49-zeta T cells did not mediate bystander killing of TAG-72-negative cells. In addition, CC49-zeta T cells not only killed FasL-positive tumor cells in vitro and in vivo, but also survived in their presence, and were immunoprotective in intraperitoneal and subcutaneous murine tumor xenograft models with TAG-72-positive human tumor cells. Finally, receptor-positive T cells were still effective in killing TAG-72-positive targets in the presence of physiological levels of soluble TAG-72, and did not induce killing of TAG-72-negative cells under the same conditions.
- For clinical practice of the invention several reports exist in the art that would guide the skilled artisan as to concentrations, cell numbers, and dosing protocols useful. While in the art CAR T cells have been utilized targeting surface tumor antigens, the main issue with this approach is the difficulty of T cells to enter tumors due to features specific to the tumor microenvironment. These include higher interstitial pressure inside the tumor compared to the surroundings, acidosis inside the tumor, and expression in the tumor of FasL which kills activated T cells. Accordingly the invention seeks to more effectively utilize CAR-macrophage cells by directly targeting them to tumor endothelium, which is in direct contact with blood and therefore not susceptible to intratumoral factors the limit efficacy of conventional T cell therapies. In other embodiments CAR-macrophage are targeting to tumor antigens.
- In one embodiment of the invention, protocols similar to Kershaw et al. are utilized with the exception that tumor endothelial antigens are targeted as opposed to conventional tumor antigens. Such tumor endothelial antigens include CD93, TEM-1, VEGFR1, and survivin. Antibodies can be made for these proteins, methodologies for which are described in U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, and 5,639,641. In one example that may be utilized as a template for clinical development, T cells with reactivity against the ovarian cancer-associated antigen alpha-folate receptor (FR) were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor gamma chain. Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1 received a dose escalation of T cells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specific T cells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells. Five patients in cohort 1 experienced some grade 3 to 4 treatment-related toxicity that was probably due to interleukin-2 administration, which could be managed using standard measures. Patients in cohort 2 experienced relatively mild side effects with grade 1 to 2 symptoms. No reduction in tumor burden was seen in any patient. Tracking 111In-labeled adoptively transferred T cells in cohort 1 revealed a lack of specific localization of T cells to tumor except in one patient where some signal was detected in a peritoneal deposit. PCR analysis showed that gene-modified T cells were present in the circulation in large numbers for the first 2 days after transfer, but these quickly declined to be barely detectable 1 month later in most patients. Similar CAR-T clinical studies have been reported for neuroblastoma, B cell malignancies, melanoma, ovarian cancer, renal cancer, mesothelioma, and head and neck cancer.
- In one embodiment of the invention, PBMCs are derived from leukapheresis and CD14 monocytes are collected by MACS. After 3 days of culture, M-CSF at 100 ng/ml plasmid encoding the chimeric CAR-macrophage recognizing tumor specific antigen and subsequently selected for gene integration by culture in G418. In another embodiment the generation of dual-specific T cells is performed, stimulation of allogeneic monocytic cells is achieved by coculture of patient PBMCs with irradiated (5,000 cGy) allogeneic donor PBMCs from cryopreserved apheresis product (mixed lymphocyte reaction). The MHC haplotype of allogeneic donors is determined before use, and donors that differed in at least four MHC class I alleles from the patient are used. Culture medium consisted of AimV medium (Invitrogen, Carlsbad, Calif.) supplemented with 5% human AB.sup.-serum (Valley Biomedical, Winchester, Va.), penicillin (50 units/mL), streptomycin (50 mg/mL; Bio Whittaker, Walkersville, Md.), amphotericin B (Fungizone, 1.25 mg/mL; Biofluids, Rockville, Md.), L-glutamine (2 mmol/L; Mediatech, Herndon, Va.), and human recombinant IL-2 (Proleukin, 300 IU/mL; Chiron). Mixed lymphocyte reaction consisted of 2.times.10.sup.6 patient monocytes and 1.times.10.sup.7 allogeneic stimulator PBMCs in 2 mL AimV per well in 24-well plates. Between 24 and 48 wells are cultured per patient for 3 days, at which time transduction is done by aspirating 1.5 mL of medium and replacing with 2.0 mL retroviral supernatant containing 300 IU/mL IL-2, 10 mmol/L HEPES, and 8.mu.g/mL polybrene (Sigma, St. Louis, Mo.) followed by covering with plastic wrap and centrifugation at 1,000.times.g for 1 hour at room temperature. After overnight culture at 37.degree. C./5% CO.sub.2, transduction is repeated on the following day, and then medium was replaced after another 24 hours. Cells are then resuspended at 1.times.10.sup.6/mL in fresh medium containing 0.5 mg/mL G418 (Invitrogen) in 175-cm.sup.2 flasks for 5 days before resuspension in media lacking G418. ‘Cells are expanded to 2.times.10.sup.9 and then restimulated with allogeneic PBMCs from the same donor to enrich for T cells specific for the donor allogeneic haplotype. Restimulation is done by incubating patient T cells (1.times.10.sup.6/mL) and stimulator PBMCs (2.times.10.sup.6/mL) in 3-liter Fenwall culture bags in AimV+additives and IL-2 (no G418). Cell numbers were adjusted to 1.times.10.sup.6/mL, and IL-2 was added every 2 days, until sufficient numbers for treatment were achieved.
- The present invention relates to a strategy of adoptive cell transfer of monocytes or DC transduced to express a chimeric antigen receptor (CAR). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., tumor endothelial antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor endothelium cellular immune activity. In one embodiment the present invention relates generally to the use of monocytes or DC cells genetically modified to stably express a desired CAR that possesses high affinity towards tumor associated endothelium. Monocytes or DC cells expressing a CAR are referred to herein as CAR-MACROPHAGE cells or CAR modified DC cells. Preferably, the cell can be genetically modified to stably express an antibody binding domain on its surface, conferring novel antigen specificity that is MHC independent. In some instances, the monocyte or DC cell is genetically modified to stably express a CAR that combines an antigen recognition domain of a specific antibody with an intracellular domain of the CD3-zeta chain or Fc.gamma.RI protein into a single chimeric protein. In another embodiment, TLR signaling molecules are engineered in the intracellular portion of the CAR, said molecules include TRIF, TRADD, and MyD99. In one embodiment, the CAR of the invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino add substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. Preferably, the transmembrane domain is the CD8a hinge domain. With respect to the cytoplasmic domain, the CAR of the invention can be designed to comprise the CD80 and/or CD86 and/or CD40L and/or OX40L signaling domain by itself or be combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. In one embodiment, the cytoplasmic domain of the CAR can be designed to further comprise the signaling domain of MyD88. For example, the cytoplasmic domain of the CAR can include but is not limited to CD80 and/or CD86 and/or CD40L and/or OX40L signaling modules and combinations thereof. In another embodiment of the invention inhibition of TGF-beta is performed either by transfection with an shRNA possessing selectively towards TGF-beta or by constructing the CAR to possess a dominant negative mutant of TGF-beta receptor. This would render the CAR-macrophage cell resistant to inhibitory activities of the tumors. Accordingly, the invention provides CAR-macrophage cells and methods of their use for adoptive therapy. In one embodiment, the CAR-macrophage cells of the invention can be generated by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-CD19, CD8a hinge and transmembrane domain, and MyD88, into the cells. The CAR-macrophage cells of the invention are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
- One skilled in the art will appreciate that these methods, compositions, and cells are and may be adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods, procedures, and devices described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the disclosure. It will be apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Those skilled in the art recognize that the aspects and embodiments of the invention set forth herein may be practiced separate from each other or in conjunction with each other. Therefore, combinations of separate embodiments are within the scope of the invention as disclosed herein. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. According to one embodiment of the present invention, the chimeric antigen receptor may include two intracellular signaling domains. For example, the chimeric antigen receptor may include a first intracellular signaling domain linked to the transmembrane domain and a second intracellular signaling domain linked to a terminal of the first intracellular signaling domain that is not linked with the transmembrane domain. According to a more specific embodiment, the first intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 (CD134), OX40 ligand (OX40L, CD252), 4-1BB (CD137), CD28, DAP10, CD3-zeta (CD3) and DAP12, and the second intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12. In this case, at least one of the first intracellular signaling domain and the second intracellular signaling domain includes the whole or a portion of OX40 ligand. For example, the chimeric antigen receptor may include a first intracellular signaling domain containing the whole or a portion of OX40 ligand and a second intracellular signaling domain containing the whole or a portion of any one selected from CD3-zeta and DAP12. Further, for example, the chimeric antigen receptor may include a first intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of CD3-zeta and DAP12 and a second intracellular signaling domain containing the whole or a portion of OX40 ligand. According to another embodiment of the present invention, the chimeric antigen receptor may include three intracellular signaling domains. For example, the chimeric antigen receptor may include: a tirst intracellular signaling domain linked to the transmembrane domain; a second intracellular signaling domain linked to a terminal of the first intracellular signaling domain that is not linked with the transmembrane domain; and a third intracellular signaling domain linked to a terminal of the second intracellular signaling domain that is not linked with the first intracellular signaling domain. According to a more specific embodiment, the first intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of 4-1BB, OX40, OX40 ligand, CD28 and DAP10, the second intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB, and the third intracellular signaling domain may include the whole or a portion of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12. In such a case, at least one of the first intracellular signaling domain, the second intracellular signaling domain and the third intracellular signaling domain may include the whole or a portion of OX40 ligand. In another aspect, the present invention may provide a chimeric antigen receptor, which includes: a first intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of CD28 and 4-1BB; a second intracellular signaling domain containing the whole or a portion of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB; and a third intracellular signaling domain containing the whole or a portion of CD3-zeta, wherein the first, second and third intracellular signaling domains are arranged in order from the cell membrane toward the inside of the cell. According to one embodiment of the present invention, the above respective domains may be directly linked to one another or may be linked by a linker. According to one embodiment of the present invention, the chimeric antigen receptor may further include: a transmembrane domain linked to the first intracellular signaling domain; a spacer domain linked to the transmembrane domain; and an extracellular domain linked to the spacer domain. In addition, the chimeric antigen receptor may further include a signal sequence linked to the extracellular domain. According to one embodiment of the present invention, the above respective domains may be directly linked to one another or may be linked by a linker.
- According to one embodiment of the present invention, the extracellular domain is a domain for specifically binding with an antibody or specifically recognizing an antigen, for example, an Fc receptor, an antigen-binding fragment of an antibody such as a single-chain variable fragment (ScFv), NK receptor (natural cytotoxicity receptor), NKG2D, 2B4 or DNAM-1, etc. Thus, in the present disclosure, the term “extracellular domain” is used with the same meanings as the “antigenic recognition site”, “antigen-binding fragment” and/or “antibody binding site.” The chimeric antigen receptor according to an embodiment of the present invention may include an Fc receptor as the extracellular domain, and therefore, can be used along with a variety of antibodies depending on cell types of cancer to be treated. According to one embodiment, the Fc receptor may include any one selected from the group consisting of CD16, CD32, CD64, CD23 and CD89, and variants thereof. According to a more specific embodiment, the Fc receptor may include CD16 or variants thereof, and most specifically, may include the whole or a portion of CD16 V158 variant (CD16V). According to another embodiment, the chimeric antigen receptor of the present invention may include, as the extracellular domain, an antigen-binding fragment of an antibody which directly recognizes the antigen without co-administration along with the antibody. According to one embodiment, the antigen-binding fragment may be an Fab fragment, F(ab′) fragment, F(ab′)2 fragment or Fv fragment. According to one embodiment of the present invention, the antibody may be any one of various types of antibodies capable of binding antigen-specifically. For example, the antibody may be one in which one light chain and one heavy chain are bonded with each other, or one in which two light chains and two heavy chains are bonded with each other. For example, when two light chains and two heavy chains are bonded with each other, the antibody may be one in which the first unit including the first light chain and the first heavy chain bonded with each other and the second unit including the second light chain and the second heavy chain bonded with each other are combined with each other. The bond may be a disulfide bond, but it is not limited thereto. According to an embodiment of the present invention, the above two units may be the same as or different from each other. For example, the first unit including the first light chain and the first heavy chain and the second unit including the second light chain and the second heavy chain may be the same as or different from each other. As such, an antibody prepared to recognize two different antigens by the first unit and the second unit, respectively, is commonly referred to as a ‘bispecific antibody’ in the related art. In addition, for example, the antibody may be one in which the above three or more units are combined with one another. The antigen-binding fragment of the present invention may be derived from various types of antibodies as described above, but it is not limited thereto.
- According to another embodiment of the present invention, the extracellular domain used herein may be a NK receptor (natural cytotoxicity receptor). According to a specific embodiment, the NK receptor may include NKp46, NKp30, NKp44, NKp80 and NKp65 receptors, but it is not limited thereto.
- According to one embodiment, the signal sequence may include the whole or a portion of CD16. According to another embodiment, the extracellular domain may include the whole or a portion of CD16 V158 variant (CD16V). According to another embodiment, the spacer domain may include the whole or a portion of any one selected from the group consisting of CD8.alpha. (CD8-alpha) and CD28. According to another embodiment, the transmembrane domain may include the whole or a portion of any one selected from the group consisting of CD8.alpha. and CD28.
- According to another aspect, the present invention provides immune cells (e.g., NK cells) to express the above-described chimeric antigen receptor according to the present invention.
- The immune cells of the present invention may exhibit toxicity to tumor cells. It is determined that the chimeric antigen receptor according to the invention exhibits specific toxicity to what types of tumor cells depending on what types of antibodies are combined with the extracellular domains. Therefore, the types of tumor cells, to which the immune cells expressing the chimeric antigen receptor according to the present invention may exhibit specific toxicity, are not particularly limited. According to one embodiment, when the immune cells (e.g., NK cells) of the present invention are used along with rituximab, the cells may exhibit toxicity to malignant lymphoma cells. For example, the malignant lymphoma cells may express CD20. Further, for example, the malignant lymphoma may be B-cell lymphoma.
- According to another aspect, the invention further provides a pharmaceutical composition for prevention or treatment of tumor or tumor metastasis, which includes the immune cells (e.g., NK cells) expressing the above-described chimeric antigen receptor according to the present invention, in the number of 2 to 7.5 times the number of tumor cells (e.g., malignant lymphoma cells) in a subject to be treated (‘treatment target’). According to one embodiment of the present invention, the number of immune cells (e.g., NK cells) included in the pharmaceutical composition of the present invention in a single dose may range from 0.75 to 10 times the number of tumor cells (e.g., malignant lymphoma cells) in the treatment target. For example, the number of the immune cells (e.g., NK cells) in a single dose may range from 2 to 7.5 times the number of tumor cells (e.g., malignant lymphoma cells) in the treatment target. In some embodiments of the invention NK cell lines are engineered to express CAR. In one embodiment the NK cell line is NK-92.
- Sequential administration of CAR-NK and CAR-macrophages are disclosed. In some embodiments addition of CAR-T cells is performed last.
Claims (20)
1. A method of treating cancer comprising administration of innate and adaptive immune cells, wherein said immune cells have been modified with a chimeric antigen receptor (CAR).
2. The method of claim 1 , wherein said innate immune cells are macrophages and/or an ex vivo population of CD14 expressing cells.
3. The method of claim 2 , wherein said macrophages are engineered to express a chimeric antigen receptor (CAR).
4. The method of claim 3 , wherein the CAR comprises (i) an extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain containing an intracellular signaling domain; and (b) a pharmaceutically acceptable carrier or excipient; thereby treating the cancer in the human subject.
5. The method of claim 3 , wherein the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain, an Fc.epsilon.R intracellular signaling domain, an Fc.gamma.R intracellular signaling domain, or a TRIF intracellular signaling domain.
6. The method of claim 3 , wherein the intracellular domain comprises two or more intracellular signaling domains.
7. The method of claim 3 , wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR4 transmembrane domain.
8. The method of claim 2 , wherein said macrophages are CD14+ cells comprises a population of CD14+/CD16+ cells.
9. The method of claim 2 , wherein said macrophages are CD14+ cells comprises a population of CD14+/CD56+ cells.
10. The method of claim 2 , wherein said macrophages comprise a population of CD14+ monocytes, a population of CD14+ macrophages or a population of CD14+ dendritic cells.
11. The method of claim 3 , wherein the method comprises (i) extracting a blood sample from the human subject; (ii) isolating monocytes from the blood sample; and (iii) transfecting the monocytes from the blood sample with the recombinant polynucleic acid with a sequence encoding a CAR; and wherein administering comprises infusing.
12. The method of claim 3 , wherein the recombinant polynucleic acid is mRNA.
13. The method of claim 3 , wherein the ex vivo population of CD14+ cells stimulates killing of cancer cells in the human subject by T cells of the human subject.
14. The method of claim 3 , wherein the intracellular domain of the CAR is capable of inducing monocytic differentiation to M1 macrophages in the human subject.
15. The method of claim 3 , wherein the ex vivo population of CD14+ cells enhances or improves effector function of a T cell in the human subject.
16. The method of claim 3 , wherein the ex vivo population of CD14+ cells directly kills cancer cells in the human subject.
17. The method of claim 3 , wherein the ex vivo population of CD14+ cells inhibits macrophage or macrophage related cells of the human subject from promoting tumor growth.
18. The method of claim 3 , wherein the ex vivo population of CD14+ cells is phagocytic.
19. The method of claim 3 , wherein the method further comprises administering M-CSF.
20. The method of claim 3 , wherein the method further comprises administering C5 component of complement.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/455,544 US20240066126A1 (en) | 2022-08-24 | 2023-08-24 | Combination therapy of solid tumors using chimeric antigen receptor cells representing adaptive and innate immunity |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263400740P | 2022-08-24 | 2022-08-24 | |
US18/455,544 US20240066126A1 (en) | 2022-08-24 | 2023-08-24 | Combination therapy of solid tumors using chimeric antigen receptor cells representing adaptive and innate immunity |
Publications (1)
Publication Number | Publication Date |
---|---|
US20240066126A1 true US20240066126A1 (en) | 2024-02-29 |
Family
ID=90001047
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US18/455,544 Pending US20240066126A1 (en) | 2022-08-24 | 2023-08-24 | Combination therapy of solid tumors using chimeric antigen receptor cells representing adaptive and innate immunity |
Country Status (1)
Country | Link |
---|---|
US (1) | US20240066126A1 (en) |
-
2023
- 2023-08-24 US US18/455,544 patent/US20240066126A1/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11918604B2 (en) | Chimeric antigen receptor dendritic cell (CAR-DC) for treatment of cancer | |
Constantino et al. | Dendritic cell-based immunotherapy: a basic review and recent advances | |
Nesbeth et al. | CD4+ T cells elicit host immune responses to MHC class II− ovarian cancer through CCL5 secretion and CD40-mediated licensing of dendritic cells | |
Baxevanis et al. | Cancer immunotherapy | |
Palucka et al. | Dendritic cells: a critical player in cancer therapy? | |
Avigan et al. | Dendritic/tumor fusion cells as cancer vaccines | |
JP2022507830A (en) | Modified cells expressing therapeutic agents and their use | |
Oth et al. | Pathogen-associated molecular patterns induced crosstalk between dendritic cells, T helper cells, and natural killer helper cells can improve dendritic cell vaccination | |
Yang et al. | Adoptive cellular therapy (ACT) for cancer treatment | |
Bastien et al. | Cellular therapy approaches harnessing the power of the immune system for personalized cancer treatment | |
JP2023538463A (en) | Methods of treating cancer and autoimmune and inflammatory diseases | |
Higham et al. | Activation of tolerogenic dendritic cells in the tumor draining lymph nodes by CD8+ T cells engineered to express CD40 ligand | |
Choi et al. | The past, present, and future of adoptive T cell therapy | |
US20230226113A1 (en) | Vaccine and Uses thereof in Cell Therapy | |
Bao et al. | Current status of leukemia cytotherapy-exploitation with immune cells | |
US20040260061A1 (en) | Continuous, normal human t-lymphocyte cell lines comprising a recombinant immune receptor with defined antigen specificity | |
AU2016243194A1 (en) | Compositions and methods of treating acute myeloid leukemia | |
US20240066126A1 (en) | Combination therapy of solid tumors using chimeric antigen receptor cells representing adaptive and innate immunity | |
Louis et al. | Cellular immunotherapy for neuroblastoma: a review of current vaccine and adoptive T cell therapeutics | |
US20230030680A1 (en) | Chimeric gmcsf-il18 receptor | |
CN117203328A (en) | Recombinant antigen presenting cells | |
Mohammadi et al. | Immunotherapy Research And Textbook 3: T-cell transfer therapy and Monoclonal antibodies | |
Leuci et al. | Ex vivo-activated MHC-unrestricted immune effectors for cancer adoptive immunotherapy | |
Yi et al. | Adoptive cell therapy of prostate cancer using female mice-derived T cells that react with prostate antigens | |
Sanchez et al. | T9 glioma cells expressing membrane-macrophage colony stimulating factor produce CD4+ T cell-associated protective immunity against T9 intracranial gliomas and systemic immunity against different syngeneic gliomas |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: REGEN BIOPHARMA, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ICHIM, THOMAS;KOOS, DAVID;LANDER, HARRY;REEL/FRAME:064733/0427 Effective date: 20230828 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |